Compositions and methods for diagnosis and treatment of hepatic cancers

ABSTRACT

The present invention relates to methods of treating liver cancer using a Notch signaling inhibitor. Compositions and methods for the treatment of liver cancers are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/373,275, filed Dec. 8, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/208,523, filed Mar. 13, 2014, issued as U.S.Pat. No. 9,550,829, which claims the benefit of U.S. ProvisionalApplication No. 61/789,475, filed Mar. 15, 2013, each of which isincorporated by reference herein in its entirety for any purpose.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 25, 2018, isnamed 2018-04-25_01146-0029-02US_SL.txt and is 112,298 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology. More specifically, the invention relates to methods oftreatment of pathological hepatic conditions, such as cancer.

BACKGROUND

Liver cancer is the fifth most common form of cancer. Each year,approximately 750,000 cases are diagnosed and about 700,000 people diefrom the disease each year, making it the third most common cause ofcancer death in the world (Ferlay et al., Int. J. Cancer 127:2893-2917(2010)). In the United States, the incidence of primary liver cancer hasbeen rising, and while some progress has been made in detecting andtreating localized disease, the five year survival rate for late stageliver cancer is still well below 10% (American-Cancer-Society. 2012.Cancer Facts & Figures 2012. Atlanta: American Cancer Society).

Established treatments for liver cancer include surgical removal of thepart of the liver containing the tumor (partial hepatectomy), livertransplantation, transcatheter arterial chemoembolization (TACE), insitu tumor destruction by various methods such as radiofrequencyablation (RFA) or cryosurgery and administration of Sorafenib. Treatmentoptions for late stage liver patients are limited. Thus, effectivetreatments of liver cancer remains a significant unmet medical need.

The role of Notch signaling in liver cancer is not well understood. Qiet al. report that Notch1 signaling inhibits growth of humanhepatocellular carcinoma cells in vitro and in vivo by inducing cellcycle arrest and apoptosis (Qi et al., Cancer Res. 63:8323 (2003)) andViatour et al. report that expression of Notch1 intracellular domaindecreased proliferation and induced apoptosis in murine and human HCCcells (Viatour et al., J. Exp. Med. 208(10):1963 (2011)). Others reportthat Notch1 small interfering RNA (siRNA) reduced cell invasion andmigration but not viability (Zhou et al. Dig. Dis. Sci.). Yet othersreport that inhibition of individual Notch pathway family members had noeffect. Taken together, the Notch pathway's role in liver cancer was notwell understood.

SUMMARY

Use of Notch2 signaling inhibitors for the treatment of patients havingor at risk of having proliferative disorders of the liver is provided.

In one aspect, methods of treating a liver cancer in an individual inneed thereof are provided, comprising the step of administering to theindividual an effective amount of a Notch2 signaling inhibitor. In someembodiments, the liver cancer is hepatocellular carcinoma, hepatoma,cholangiocarcinoma, hepatoblastoma, hepatic carcinoma, hepaticangiosarcoma, or metastatic liver cancer. In some embodiments, thehepatocellular carcinoma comprises progenitor-like orcholangiocarcinoma-like liver tumor. In some embodiments, the livercancer is a refractory cancer.

In some embodiments, the method further comprises administering at leastone additional therapeutic agent. Examples of additional therapeuticagents include, but are not limited to chemotherapeutic agent and anantibody.

In some embodiments, the liver cancer comprises cells that expressEpCAM, AFP, Notch2, Jag1, Notch2 and Jag1, nuclear Notch2 ICD, Sox9,CK19, Ras, Prom1, Spp1, FoxM1, Plk1, ccnb1, Aurkb, Wnt2, Axin2, or Glu1,or any combination thereof. In specific embodiments, the liver cancercomprises cells that are AFP⁺ EpCAM⁺. In some embodiments, the livercancer comprises cells that are AFP⁺ EpCAM⁻, AFP⁺ EpCAM⁺ SPP1⁺, AFP⁻EpCAM⁺, AFP⁻ EpCAM⁺ Notch2⁺, AFP⁺ EpCAM⁻ Notch2⁺, AFP⁺ EpCAM⁺ Sox9⁺ andAFP⁺ EpCAM⁺ Sox9⁺ or AFP⁻ EpCAM⁺ SPP1⁺. Liver cancers that comprisecells with alternative combinations of marker expression arespecifically contemplated.

In some embodiments, at least one of EpCAM, AFP, Notch2, Jag1, Sox9,CK19, Ras, Prom1, Spp1, FoxM1, Plk1, ccnb1, Aurkb, Wnt2, Axin2, or Glu1protein expression was determined in a sample from the individual usingimmunohistochemistry (IHC). In some embodiments, expression is nucleicacid expression. In some embodiments, expression is determined by amethod selected from the group consisting of RNAseq, microarrayanalysis, immunohistochemistry, enzyme-linked immunosorbent assay, geneexpression profiling, polymerase chain reaction, SAGE, MassARRAYtechnique, fluorescent in situ hybridization and Western blotting.

In some embodiments, administering the Notch2 signaling inhibitorresults in a decrease in the expression in the liver cancer of at leastone of EpCAM, AFP, Notch2, Notch2 ICD, Jag1, Prom1, Spp1, FoxM1, Plk1,ccnb1 and Aurkb. In some embodiments, administering the Notch2 signalinginhibitor results in an increase in the expression in the liver cancerof at least one of Wnt2, Axin2 and Glu1. In some embodiments, expressionis determined by RNAseq, microarray analysis, immunohistochemistry,enzyme-linked immunosorbent assay, and Western blotting.

Any of the antibodies of the above embodiments may be a full-length IgG1or IgG2a antibody. In some embodiments, the antibody causes cancer celldeath, e.g., liver cancer cell death. Any of the antibodies in the aboveembodiments may be conjugated to a growth inhibitory agent, e.g., acytotoxic agent. Examples of cytotoxic agent include, but are notlimited to, toxins, antibiotics, radioactive isotopes and nucleolyticenzymes. Any of the antibodies in the above embodiments may be producedby known methods in the art, e.g., in bacteria or in CHO cells.

In one aspect, methods are provided for preventing liver cancer in anindividual at risk of having liver cancer, comprising the step ofadministering to the individual an effective amount of a Notch2signaling inhibitor. In some embodiments, the individual has a livercondition selected from the group consisting of hepatitis B or C,cirrhosis of the liver, non-viral/non-alcoholic steatohepatitis, benignliver tumors, hemangiomas, hepatic adenomas, and focal nodularhyperplasia. In some embodiments, the Notch2 signaling inhibitor is ananti-Jag1 antibody, e.g., an anti-Jag1 antagonist antibody.

In one aspect, methods are provided for inhibiting growth of a cell thatexpresses secreted phosphoprotein1 (SPP1), comprising contacting thecell with a Notch2 signaling inhibitor, thereby inhibiting growth of thecell. In one embodiment, SPP1 protein comprises the amino acid sequenceshown in FIG. 11. In one embodiment, contacting the cell with the Notch2signaling inhibitor reduces SPP1 expression in the cell. For example,contacting the cell with the Notch2 signaling inhibitor reduces SPP1expression in the cell by at least about 50%, 60%, 70%, 80%, 90%, or100%. The expression of SPP1 mRNA or protein can be determined by anymethod in the art. In some embodiments, the Notch2 signaling inhibitoris an anti-Notch2 antibody, e.g., an anti-Notch2 negative regulatoryregion (NRR) antibody, such as any anti-Notch2 NRR antibody disclosedherein. In some embodiments, the antibody is an anti-Jag1 antibody, suchas any anti-Jag1 antibody disclosed herein. In some embodiments, thecell is a liver cancer cell. In some embodiments, the liver cancer cellexpresses EpCAM, AFP, AFP and EpCAM, Notch2, Jag1, Notch2 and Jag1,nuclear Notch2 ICD, Ras, Prom1, Spp1, FoxM1, Plk1, ccnb1, Aurkb, Wnt2,Axin2, or Glu1, or any combination thereof. In some embodiments,contacting the cell with the Notch2 signaling inhibitor results in adecrease in the expression in the cell of at least one of EpCAM, AFP,Notch2, Notch2 ICD, Jag1, Prom1, Spp1, FoxM1, Plk1, ccnb1 and Aurkb. Insome embodiments, administering the Notch2 signaling inhibitor resultsin an increase in the expression in the cell of at least one of Wnt2,Axin2 and Glu1. In some embodiments, expression is determined by RNAseq,microarray analysis, immunohistochemistry, enzyme-linked immunosorbentassay, and Western blotting.

In one aspect, methods are provided for inhibiting proliferation of acell that expresses secreted phosphoprotein1 (SPP1), comprisingcontacting the cell with a Notch2 signaling inhibitor, therebyinhibiting proliferation of the cell.

In one aspect, methods are provided for treating a mammal having a livercancer comprising cells that express a Spp1 gene encoding a peptidecomprising an amino acid sequence having at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the polypeptideshown in FIG. 11, comprising administering to the mammal an effectiveamount of a Notch2 signaling inhibitor, thereby effectively treating themammal. In some embodiments, the cells express a SPP1 protein comprisingan amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100% identity to the polypeptide shown in FIG.11.

In some embodiments, the liver cancer is hepatocellular carcinoma,hepatoma, cholangiocarcinoma, hepatoblastoma, hepatic carcinoma, hepaticangiosarcoma, or metastatic liver cancer. In some embodiments, the livercancer is a refractory cancer. Any of the antibodies herein may be afull-length IgG1 or IgG2a antibody. In some embodiments, the antibodycauses cancer cell death, e.g., liver cell death. Any of the herein maybe conjugated to a growth inhibitory agent, e.g., a cytotoxic agent.Examples of cytotoxic agent include, but are not limited to, toxins,antibiotics, radioactive isotopes and nucleolytic enzymes. Any of theantibodies herein may be produced by known methods in the art, e.g., inbacteria or in CHO cells.

In some embodiments, the method further comprises administering at leastone additional therapeutic agent. Examples of additional therapeuticagents include, but are not limited to chemotherapeutic agent and anantibody.

In some embodiments, the liver cancer comprises cells that expressEpCAM, AFP, AFP and EpCAM, Notch2, Jag1, Notch2 and Jag1, nuclear Notch2ICD, Ras, Prom1, Spp1, FoxM1, Plk1, ccnb1, Aurkb, Wnt2, Axin2, or Glu1,or any combination thereof. In some embodiments, administering theNotch2 signaling inhibitor results in a decrease in the expression inthe liver cancer of at least one of EpCAM, AFP, Notch2, Notch2 ICD,Jag1, Prom1, Spp1, FoxM1, Plk1, ccnb1 and Aurkb. In some embodiments,administering the Notch2 signaling inhibitor results in an increase inthe expression in the liver cancer of at least one of Wnt2, Axin2 andGlu1. In some embodiments, expression is determined by RNAseq,microarray analysis, immunohistochemistry, enzyme-linked immunosorbentassay, and Western blotting.

In one aspect, methods are provided for treating a liver cellproliferative disorder associated with increased expression or activityof a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100% amino acid sequence identity to the polypeptide shown inFIG. 8C, comprising administering to an individual in need of suchtreatment an effective amount of an anti-Jag1 antagonist antibody,thereby effectively treating the liver cell proliferative disorder. Insome embodiments, the cell proliferative disorder is a cancer, such asliver cancer. In some embodiments the individual has a liver conditionselected from the group consisting of hepatitis B or C, cirrhosis of theliver, benign liver tumors, hemangiomas, hepatic adenomas, and focalnodular hyperplasia.

In certain embodiments, the anti-Jag1 antibody is any of the anti-Jag1antibodies described herein. In certain embodiments, the anti-Jag1antibody is a human, humanized, or chimeric antibody. In certainembodiments, any of the antibodies of the above embodiments is anantibody fragment.

In one aspect, methods are provided for treating a liver cellproliferative disorder associated with increased expression or activityof a protein having at least 90% amino acid sequence identity to thepolypeptide shown in FIG. 11, comprising administering to an individualin need of such treatment an effective amount of an anti-Jag1 antagonistantibody, thereby effectively treating the liver cell proliferativedisorder. In some embodiments, the cell proliferative disorder is acancer, such as liver cancer. In some embodiments the individual has aliver condition selected from the group consisting of hepatitis B or C,cirrhosis of the liver, benign liver tumors, hemangiomas, hepaticadenomas, and focal nodular hyperplasia.

In one aspect, methods are provided for reducing serum SPP1 proteinlevels in an individual, the method comprising administering to theindividual an effective amount of a Notch2 signaling inhibitor therebyreducing serum SPP1 levels in the individual. In one embodiment, theindividual has a liver cancer. In one embodiment, the serum SPP1 proteinlevels prior to administering the Notch2 signaling inhibitor to theindividual are at least about 80 ng/ml. In certain embodiments, theserum SPP1 protein levels prior to administering the Notch2 signalinginhibitor to the individual are between about 80 ng/ml and about 500ng/ml, between about 86 ng/ml and about 250 ng/ml, between about 120ng/ml and about 170 ng/ml, or about 165 ng/ml. In some embodiments,administering the Notch2 signaling inhibitor to the individual resultsin serum SPP1 protein levels of less than 80 ng/ml. In specificembodiments, serum SPP1 protein levels prior to administering the Notch2signaling inhibitor are 24 hours prior to administering the Notch2signaling inhibitor. Serum SPP1 protein levels prior to or followingadministration of the Notch2 signaling inhibitor may be determined byany appropriate method, such as enzyme-linked immunosorbent assay. Inspecific embodiments, serum SPP1 protein levels are reduced about one,two, three, six or 12 month after administering the Notch2 signalinginhibitor. In some embodiments, the liver cancer is hepatocellularcarcinoma, hepatoma, cholangiocarcinoma, hepatoblastoma, hepaticcarcinoma, hepatic angiosarcoma or metastatic liver cancer. In someembodiments, the Notch2 signaling inhibitor is an siRNA, small-moleculeinhibitor or antibody. In some embodiments, the antibody is anantagonist antibody.

In certain embodiments, any of the antibodies of the above embodimentsis a monoclonal antibody. In certain embodiments, any of the antibodiesof the above embodiments is a human, humanized, or chimeric antibody. Incertain embodiments, any of the antibodies of the above embodiments isan antibody fragment.

In some embodiments, the method further comprises administering at leastone additional therapeutic agent. Examples of additional therapeuticagents include, but are not limited to chemotherapeutic agent and anantibody. In some embodiments, Sorafenib is an additional therapeuticagent.

In some embodiments, the liver cancer comprises cells that expressEpCAM, AFP, AFP and EpCAM, Notch2, Jag1, Notch2 and Jag1, nuclear Notch2ICD, Ras, Prom1, Spp1, FoxM1, Plk1, ccnb1, Aurkb, Wnt2, Axin2, or Glu1,or any combination thereof. In some embodiments, Ras is a mutant Ras. Insome embodiments, administering the Notch2 signaling inhibitor resultsin a decrease in the expression in the liver cancer of at least one ofEpCAM, AFP, Notch2, Notch2 ICD, Jag1, Prom1, Spp1, FoxM1, Plk1, ccnb1and Aurkb. In some embodiments, administering the Notch2 signalinginhibitor results in an increase in the expression in the liver cancerof at least one of Wnt2, Axin2 and Glu1. In some embodiments, expressionis determined by RNAseq, microarray analysis, immunohistochemistry,enzyme-linked immunosorbent assay, and Western blotting.

In one aspect, methods are provided for treating a liver tumor in amammal, wherein the growth of the liver tumor is at least in partdependent upon a growth potentiating effect of Notch2 signaling,comprising contacting the tumor with an antibody that binds to Notch2 orJag1. In one embodiment, binding of the antibody to the tumorantagonizes the growth-potentiating activity of Notch2. In someembodiments, the mammal is a human.

In one aspect, methods are provided for treating of liver cancercomprising administering to an individual who has elevated serum SPP1protein levels an effective amount of a Notch2 signaling inhibitor. Inone embodiment, the serum SPP1 protein levels of the individual are atleast about 80 ng/ml. In certain embodiments, the serum SPP1 proteinlevels prior to administering the Notch2 signaling inhibitor to theindividual are between about 80 ng/ml and about 500 ng/ml, between about86 ng/ml and about 250 ng/ml, between about 120 ng/ml and about 170ng/ml, or about 165 ng/ml. In some embodiments, administering the Notch2signaling inhibitor to the individual results in serum SPP1 proteinlevels of less than 80 ng/ml. In specific embodiments, serum SPP1protein levels prior to administering the Notch2 signaling inhibitor are24 hours prior to administering the Notch2 signaling inhibitor. SerumSPP1 protein levels prior to or following administration of the Notch2signaling inhibitor may be determined by any appropriate method, such asenzyme-linked immunosorbent assay. In specific embodiments, serum SPP1protein levels are reduced in the individual about one, two, three, sixor 12 month after administering the Notch2 signaling inhibitor. In someembodiments, the liver cancer is hepatocellular carcinoma, hepatoma,cholangiocarcinoma, hepatoblastoma, hepatic carcinoma, hepaticangiosarcoma or metastatic liver cancer. In some embodiments, the Notch2signaling inhibitor is an siRNA, small-molecule inhibitor or antibody.In some embodiments, the antibody is an antagonist antibody, such as ananti-Notch2 antagonist antibody or an anti-Jag1 antagonist antibody.

In some aspects, methods are provided for treating an individual havinga liver cancer, comprising the steps of administering to the individuala Notch2 signaling inhibitor; and determining Notch2 signaling, whereina decrease in Notch2 signaling following treatment, compared to Notch2signaling prior to treatment, is indicative of reduction of liver cancerin the individual. In some embodiments, Notch2 signaling is determinedby measuring Notch2 ICD nuclear localization, e.g., byimmunohistochemical analysis of a liver cancer sample from theindividual. In some embodiments, Notch2 signaling is determined bymeasuring expression of a gene selected from the group consisting ofNotch2, Jag1, Hes and Hey1. Expression can be determined by any method,e.g., RT-PCR, microarray, and RNAseq analysis. In some embodiments, theliver cancer is hepatocellular carcinoma, hepatoma, cholangiocarcinoma,hepatoblastoma, hepatic carcinoma, hepatic angiosarcoma, and metastaticliver cancer. In some embodiments, the Notch2 signaling inhibitor is ansiRNA, small-molecule inhibitor or antibody. In some embodiments, theantibody is an antagonist antibody, such as an anti-Notch2 antagonistantibody or an anti-Jag1 antagonist antibody.

In some aspects, methods for inhibiting cellular proliferationcomprising treating mammalian liver cancer cells with an antibody toNotch2 or Jag1, whereby proliferation of the liver cancer cell isinhibited. In certain embodiments, the cell is in a patient. In certainembodiments, the cell is in a culture medium. In certain embodiments,the cell is a liver cancer cell. In certain embodiments, the antibody isan anti-Notch2 or anti-Jag1 antagonist antibody is as described herein.In certain embodiments, the antibody is a human, humanized, or chimericantibody. In certain embodiments, any of the antibodies of the aboveembodiments is an antibody fragment.

In any of the methods herein, Notch2 signaling inhibitors may be thefollowing inhibitors. In some embodiments, the Notch2 signalinginhibitor is a siRNA, small-molecule inhibitor or antibody. In someembodiments, the antibody is an antagonist antibody. In someembodiments, the antibody is an anti-Notch2 antibody, e.g., ananti-Notch2 negative regulatory region (NRR) antibody. In someembodiments, the antibody does not significantly bind to a Notch familymember other than Notch2. In some embodiments, the antibody binds tomouse Notch2 NRR and human Notch2 NRR, e.g., with a Kd of ≤10 nM. Insome embodiments, the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4;    -   (d) an HVR-L1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:9;    -   (e) an HVR-L2 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:14; and    -   (f) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.

In further embodiments, the antibody is an anti-Jag1 antibody. In someembodiments, the antibody comprises at least one, two, three, four,five, or six HVRs selected from: (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:81; (b) HVR-H2 comprising an amino acid sequenceof SEQ ID NO:84; (c) HVR-H3 comprising an amino acid sequence of SEQ IDNO:87; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:88;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:89; and (f)HVR-L3 comprising an amino acid sequence of SEQ ID NO:92. In oneembodiment, the antibody comprises: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO:81; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO:82; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:85; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO:88; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:89; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:90.

In one embodiment, the antibody comprises: (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO:81; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO:82; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:86; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO:88; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:89; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:91. In one embodiment, the antibody comprises: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:83; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:85; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:88; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:89; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:90.

In certain embodiments, any of the antibodies of the embodiments hereinis a monoclonal antibody. In certain embodiments, any of the antibodiesof the above embodiments is a human, humanized, or chimeric antibody. Incertain embodiments, any of the antibodies of the above embodiments isan antibody fragment.

In a further embodiment, any antibody of the embodiments herein furthercomprises light chain variable domain framework LC-FR1, LC-FR2, LC-FR3,and LC-FR4 comprising, in order, the amino acid sequence of huMAb4D5-8light chain variable domain framework LC-FR1, LC-FR2, LC-FR3, and LC-FR4of FIG. 18.

In a further embodiment, any antibody of the embodiments herein furthercomprises a heavy chain variable domain framework HC-FR1, HC-FR2,HC-FR3, and HC-FR4 comprising, in order, the amino acid sequence ofhuMAb4D5-8 heavy chain variable domain framework HC-FR1, HC-FR2, HC-FR3,and HC-FR4 of FIG. 18.

In a further embodiment, any antibody of the embodiments herein furthercomprises a light chain variable domain framework LC-FR1, LC-FR2,LC-FR3, and LC-FR4 comprising, in order, the amino acid sequence ofhuMAb4D5-8 light chain variable domain framework LC-FR1, LC-FR2, LC-FR3,and LC-FR4 of FIG. 19.

In a further embodiment, any antibody of the embodiments herein furthercomprises a heavy chain variable domain framework HC-FR1, HC-FR2,HC-FR3, and HC-FR4 comprising, in order, the amino acid sequence ofhuMAb4D5-8 heavy chain variable domain framework HC-FR1, HC-FR2, HC-FR3,and HC-FR4 of FIG. 19.

In certain embodiments, the antibody is an isolated antibody that bindsto Jag1, comprising (a) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:94; (b) a VL sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO:97; or (c) a VH sequence as in (a) and a VL sequence as in (b). Insome embodiments, the antibody comprises a VH sequence of SEQ ID NO:94.In some embodiments, the antibody comprises a VL sequence of SEQ IDNO:97. In some embodiments, the antibody comprises a VH sequence of SEQID NO:94 and a VL sequence of SEQ ID NO:97. In some embodiments, theantibody comprises (a) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:95; (b) a VL sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO:98; or (c) a VH sequence as in (a) and a VL sequence as in (b). Insome embodiments, the antibody comprises a VH sequence of SEQ ID NO:95.In some embodiments, the antibody comprises a VL sequence of SEQ IDNO:98. In some embodiments, the antibody comprises a VH sequence of SEQID NO:95 and a VL sequence of SEQ ID NO:98.

In certain embodiments, the antibody is an isolated antibody that bindsto Jag1, comprising (a) a VH sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:93; (b) a VL sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO:96; or (c) a VH sequence as in (a) and a VL sequence as in (b). Insome embodiments, the antibody comprises a VH sequence of SEQ ID NO:93.In some embodiments, the antibody comprises a VL sequence of SEQ IDNO:96. In some embodiments, the antibody comprises a VH sequence of SEQID NO:93 and a VL sequence of SEQ ID NO:96.

In one aspect, an article of manufacture is provided comprising (a) acontainer; (b) a composition of matter contained within the containercomprising an anti-Notch2 antibody or an anti-Jagged1 antibody and acarrier for the treatment of liver cancer; and (c) a label affixed tothe container, or a package insert included with the container,referring to the use of the composition of matter for the therapeutictreatment of or the diagnostic detection of a liver cancer.

In one aspect, an anti-Notch2 antibody for use in the treatment of aliver cancer is provided. In certain embodiments, the liver cancer ishepatocellular carcinoma. In certain embodiments, the antibody is ananti-Notch2 NRR antagonist antibody. In one aspect, an anti-Jag1antibody for use in the treatment of a liver cancer is provided. Incertain embodiments, the liver cancer is hepatocellular carcinoma. Incertain embodiments, the antibody is an anti-Jag1 antagonist antibody.

In one aspect, use of an anti-Notch2 antibody in the preparation of amedicament for the therapeutic treatment of a liver cancer is provided.In one aspect, use of an anti-Jagged1 antibody in the preparation of amedicament for the therapeutic treatment of a liver cancer is provided.

In one aspect, use of an article of manufacture described herein in thepreparation of a medicament for the therapeutic treatment of a livercancer is provided. In one aspect, use of an article of manufacture asdescribed herein in the preparation of a medicament for treatment orprevention of a liver cell proliferative disorder is provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-F illustrate characterization of liver cancer marker expressionin the AKT/Ras HTV liver cancer model. FIGS. 1A-C depictimmunohistochemical staining of tumors that are AFP⁺EpCAM⁻ (FIG. 1A),AFP⁺EpCAM⁺ (FIG. 1B) and AFP⁻EpCAM⁺ (FIG. 1C). FIG. 1D depicts theprevalence of marker expression, expressed in percentage of total cells.FIG. 1F depicts immunohistochemical staining of activation of Notch2signaling indicated by localization of the Notch2 protein in thenucleus. FIG. 1E depicts the percent cells with nuclear Notch2 staining.

FIGS. 2A-D illustrate reduced tumor burden in Ras/AKT HTV mice treatedwith anti-Notch2 or anti-Jag1 antagonist antibodies. FIG. 2A depictsisolated livers from HTV mice treated with an isotype control antibody(upper left), an anti-Notch2 antibody (upper right) or an anti-Jag1antibody (lower left) beginning on the day of the HTV. FIG. 2B depictsRas/AKT HTV mice liver weight following antibody treatment administeredat the time of HTV injection, expressed in percent body weight. FIG. 2Cdepicts Ras/AKT HTV mice liver weight following antibody treatmentadministered two weeks after HTV injection, expressed in percent bodyweight (p<0.05, n>8). FIG. 2D depicts Ras/AKT HTV mice liver weightfollowing antibody treatment (p<0.02, n>6).

FIGS. 3A-H illustrate treatment of AKT/Ras HTV tumor-bearing mice withNotch inhibitory antibodies impedes the development of a broad range oftumor types. FIG. 3A depicts immunofluorescence analysis of AFP andEpCAM expression in livers of AKT/Ras HTV mice treated with anti-Notch2,anti-Jag1 or isotype control antibody. FIGS. 3B and C depict a decreasein EpCAM⁺ (FIG. 3B; p<0.007, n≥7) and AFP⁺ (FIG. 3C; p<0.03, n≥7) areafollowing anti-Notch2 and anti-Jag1 treatment. FIG. 3D depictsimmunofluorescence analysis of AFP and EpCAM expression in livers ofAKT/Ras HTV mice treated with anti-Notch1 antibody. FIG. 3E depicts thepercentage of EpCAM⁺ cells following AKT/Ras HTV mice treatment with ananti-Notch1, anti-Notch2, anti-Notch3, anti-Jag1 or isotype controlantibody. FIG. 3F depicts relative expression of Cytokeratin 19 (CK19)following AKT/Ras HTV mice treatment with an anti-Notch1, anti-Notch2,anti-Notch3, anti-Jag1 or isotype control antibody. FIG. 3G depicts RNAand FIG. 3H depicts protein levels of Sox9 following AKT/Ras HTV micetreatment with an anti-Notch1, anti-Notch2, anti-Notch3, anti-Jag1 orisotype control antibody.

FIGS. 4A-E illustrate that treatment of AKT/Ras HTV mice with Notchinhibitory antibodies reduces Notch pathway activation in tumor-bearinglivers. FIG. 4A depicts Notch2 nuclear immunohistochemical stainingexpressed in number of cells. FIG. 4B depicts relative expression ofNotch2, determined by QRTPCR. FIG. 4C depicts immunohistochemicalstaining for Hes1 in AKT/Ras HTV tumor-bearing livers. FIG. 4D depictsthe fraction of Hes1⁺ cells, determined by immunohistochemistry. FIG. 4Edepicts relative expression of HeyL, determined by QRTPCR.

FIGS. 5A-I illustrate the effect of Notch inhibitory antibodies onexpression of Notch signaling pathway components in the AKT/Ras model ofliver cancer. Mice subjected to AKT/Ras HTV were treated withantagonistic antibodies to Notch1, Notch2, Notch3, or Jag1, or ananti-Ragweed negative control antibody and quantitative real-time PCRwas performed on the isolated RNA from livers after 5 weeks for Notch1(FIG. 5A), Notch2 (FIG. 5B), Notch3 (FIG. 5C), Notch4 (FIG. 5D), Jag1(FIG. 5E), Jag2 (FIG. 5F), DLL1 (FIG. 5G), DLL3 (FIG. 5H), DLL4 (FIG.5I).

FIGS. 6A-I illustrate results from RNAseq analysis of livers fromAKT/Ras HTV mice treated with isotype control, anti-Notch2 or anti-Jag1antibody. Depicted are normalized counts for Prom1 (FIG. 6A), Spp1 (FIG.6B), FoxM1 (FIG. 6C), Plk1 (FIG. 6D), ccnb1 (FIG. 6E), Aurkb (FIG. 6F),Wnt2 (FIG. 6G), Axin2 (FIG. 6H) and glutamine synthetase (Glu1, FIG.6I).

FIGS. 7A-B illustrate expression of Notch2 in human HCC. FIG. 7A depictsNotch1, Notch2 and Notch3 expression, determined by RT-PCR, in culturedhuman HCC cell lines. FIG. 7B depicts immunohistochemical staining ofhuman HCC tumors for Notch2, Jag1 and Hes1.

FIGS. 8A-D show exemplary amino acid sequences of human (C) and murine(D) Notch2 protein and human (A) and murine (B) Notch2 negativeregulatory region (NRR).

FIG. 9 shows exemplary amino acid sequences of human and murine Jagged 1protein.

FIGS. 10A-B show the amino acid sequences of peptides used for phageantibody library screening and selection. All proteins were expressed asa secreted protein in BEVS cells and their sequences are listed in theN-terminal to C-terminal direction. FIG. 10A shows the amino acidsequence of expressed protein murine Jagged 1-DSL-EGF1-4 (Q34-D377). Thebold font at the N-terminus represents a short linker sequence (ADLGS(SEQ ID NO: 2)). The bold font at the C-terminus represents a shortlinker sequence (EFG), a thrombin cleavage site (LVPRGS (SEQ ID NO:26)), a G spacer and the 6-His tag (SEQ ID NO: 27). FIG. 10B shows theamino acid sequence of expressed protein human Jag1-DSL-EGF1-4. Only theJag1 sequence is shown although the antigen also contained a TEVprotease cleavage site and 6-His tag (SEQ ID NO: 27) at the C-terminus.

FIG. 11 shows an exemplary amino acid sequences of human secretedphosphoprotein1 (SPP1).

FIG. 12 shows the H1, H2, and H3 heavy chain hypervariable region (HVR)sequences of anti-Notch2 NRR antagonist antibodies. Amino acid positionsare numbered according to the Kabat numbering system as described below.

FIG. 13 shows the L1, L2, and L3 light chain HVR sequences ofanti-Notch2 NRR antagonist antibodies. Amino acid positions are numberedaccording to the Kabat numbering system as described below.

FIGS. 14A-B show an alignment of the amino acid sequences for the heavychain variable domains of anti-Notch2 antibodies. Amino acid positionsof the complementarity determining regions (CDRs) are indicated.

FIGS. 15A-B show an alignment of the amino acid sequences for the lightchain variable domains of anti-Notch2 antibodies. Amino acid positionsof the complementarity determining regions (CDRs) are indicated.

FIGS. 16A-B show exemplary acceptor human variable heavy (VH) consensusframework sequences for use in practicing the instant invention.Sequence identifiers are as follows:

-   -   human VH subgroup I consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:32, 33, 34, 35).    -   human VH subgroup I consensus frameworks “B,” “C,” and “D” minus        extended hypervariable regions (SEQ ID NOs:36, 37, 34, 35; SEQ        ID NOs:36, 37, 38, 35; and SEQ ID NOs:36, 37, 39, 35).    -   human VH subgroup II consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:40, 41, 42, 35).    -   human VH subgroup II consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:43, 44, 42, 35;        SEQ ID NOs:43, 44, 45, 35; and SEQ ID NOs:43, 44, 46, and 35).    -   human VH subgroup III consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:47, 48, 49, 35).    -   human VH subgroup III consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:50, 51, 49, 35;        SEQ ID NOs:50, 51, 52, 35; and SEQ ID NOs:50, 51, 53, 35).    -   human VH acceptor framework “A” minus Kabat CDRs (SEQ ID NOs:54,        48, 55, 35).    -   human VH acceptor frameworks “B” and “C” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 55, 35; and SEQ ID        NOs:50, 51, 56, 35).    -   human VH acceptor 2 framework “A” minus Kabat CDRs (SEQ ID        NOs:54, 48, 57, 35).    -   human VH acceptor 2 framework “B,” “C,” and “D” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 57, 35; SEQ ID NOs:50,        51, 58, 35; and SEQ ID NOs:50, 51, 59, 35).

FIG. 17 shows exemplary acceptor human variable light (VL) consensusframework sequences for use in practicing the instant invention.Sequence identifiers are as follows:

-   -   human VL kappa subgroup I consensus framework (κv1): SEQ ID        NOs:60, 61, 62, 63    -   human VL kappa subgroup II consensus framework (κv2): SEQ ID        NOs:64, 65, 66, 63    -   human VL kappa subgroup III consensus framework (κv3): SEQ ID        NOs:67, 68, 69, 63    -   human VL kappa subgroup IV consensus framework (κv4): SEQ ID        NOs:70, 71, 72, 63.

FIG. 18 depicts framework region sequences of huMAb4D5-8 light and heavychains (SEQ ID NOS 60, 61, 30, 63, 50, 51, 59 and 35, respectively, inorder of appearance). Numbers in superscript indicate amino acidpositions according to Kabat.

FIG. 19 depicts modified/variant framework region sequences ofhuMAb4D5-8 light and heavy chains (SEQ ID NOS 60, 61, 62, 31, 50, 51, 53and 35, respectively, in order of appearance). Numbers in superscriptindicate amino acid positions according to Kabat.

FIG. 20 shows the H1, H2, and H3 heavy chain hypervariable region (HVR)sequences of anti-Jagged antibodies, as described in the Examples. Aminoacid positions are numbered according to the Kabat numbering system asdescribed below.

FIG. 21 shows the L1, L2, and L3 light chain HVR sequences ofanti-Jagged antibodies, as described in the Examples. Amino acidpositions are numbered according to the Kabat numbering system asdescribed below.

FIG. 22 shows light and heavy chain variable domain framework sequencesof anti-Jagged antibodies (SEQ ID NOS 60, 61, 62, 77, 50, 99, 57 and 35,respectively, in order of appearance). Numbers in superscript indicateamino acid positions according to Kabat.

FIGS. 23A-B show an alignment of the amino acid sequences for the heavychain variable domains of antibodies to Notch2 (SEQ ID NO: 100) (B?),Notch1 (SEQ ID NO: 101) (Y), Notch3 (SEQ ID NO: 102) (W) and Jag1 (SEQID NO: 103) (A-2) described in the Examples.

FIGS. 24A-B show an alignment of the amino acid sequences for the lightchain variable domains of antibodies to Notch2 (SEQ ID NO: 104), Notch1(SEQ ID NO: 105), Notch3 (SEQ ID NO: 106) and Jag1 (SEQ ID NO: 107) usedin the Examples.

FIGS. 25A-B show an alignment of the amino acid sequences for the heavy(FIG. 25A) and light (FIG. 25B) chain variable domains of anti-Jag1antibodies. Amino acid positions of the complementarity determiningregions (CDRs) are indicated.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-Jag1 antibody” and “an antibody that binds to Jag1”refer to an antibody that is capable of binding Jag1 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting Jag1. In one embodiment, the extent ofbinding of an anti Jag1 antibody to an unrelated, non-Jag1 protein isless than about 10% of the binding of the antibody to Jag1 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to Jag1 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-Jag1 antibody binds to an epitope of Jag1 that isconserved among Jag1 from different species.

An “anti-Jag1 antagonist antibody” is an anti-Jag1 antibody that effectsdecreased Jag1-mediated signaling, e.g., Jag1-mediated Notch2 signaling.

The terms “anti-Notch2 antibody” and “an antibody that binds to Notch2”refer to an antibody that is capable of binding Notch2 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting Notch2. In one embodiment, the extent ofbinding of an anti-Notch2 antibody to an unrelated, non-Notch2 proteinis less than about 10% of the binding of the antibody to Notch2 asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that binds to Notch2 has a dissociation constant (Kd) of ≤1 μM,≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Incertain embodiments, an anti-Notch2 antibody binds to an epitope ofNotch2 that is conserved among Notch2 from different species.

An “anti-Notch2 antagonist antibody” is an anti-Notch2 antibody(including an anti-Notch2 NRR antibody) that effects decreased Notch2signaling, as defined below.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

A “blocking” antibody or an “antagonist” antibody is one whichsignificantly inhibits (either partially or completely) a biologicalactivity of the antigen it binds.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of liver cancer include,but are not limited to, hepatocellular carcinoma, hepatoma,hepatoblastoma, cholangiocarcinoma, hepatoblastoma, hepatic carcinoma,sarcoma, lymphoma and hepatic angiosarcoma. Liver cancer also includescancer that originated in the liver and has metastasized to another partof the body.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

A “chemotherapeutic agent” refers to a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (Sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®,Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib),proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors (see definition below);serine-threonine kinase inhibitors such as rapamycin (sirolimus,RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,SARASAR™); and pharmaceutically acceptable salts, acids or derivativesof any of the above; as well as combinations of two or more of the abovesuch as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; luteinizing hormone-releasing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and triptorelin; sex steroids, including progestins such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretionic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g. At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹², and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

The term “cytostatic agent” refers to a compound or composition whicharrests growth of a cell either in vitro or in vivo. Thus, a cytostaticagent may be one which significantly reduces the percentage of cells inS phase. Further examples of cytostatic agents include agents that blockcell cycle progression by inducing G0/G1 arrest or M-phase arrest. Thehumanized anti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of acytostatic agent that induces G0/G1 arrest. Classical M-phase blockersinclude the vincas (vincristine and vinblastine), taxanes, andtopoisomerase II inhibitors such as doxorubicin, epirubicin,daunorubicin, etoposide, and bleomycin. Certain agents that arrest G1also spill over into S-phase arrest, for example, DNA alkylating agentssuch as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in Mendelsohn and Israel, eds., The Molecular Basis of Cancer,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W.B. Saunders, Philadelphia,1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) areanticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®,Rhone-Poulenc Rorer), derived from the European yew, is a semisyntheticanalogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel anddocetaxel promote the assembly of microtubules from tubulin dimers andstabilize microtubules by preventing depolymerization, which results inthe inhibition of mitosis in cells.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “expression” refers to conversion of the information encoded ina gene into messenger RNA (mRNA), and then to the protein.

Herein, a sample or cell that “expresses” a protein of interest is onein which mRNA encoding the protein, or the protein, including fragmentsthereof, is determined to be present in the sample or cell.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

In one embodiment, HVR residues comprise those identified in FIGS. 12,13, 20 and 21.

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “individual at risk of having liver cancer” to an individual having ahigher than average propensity of acquiring liver cancer. Examples ofindividuals at risk of having liver cancer include, without limitation,individuals having hepatitis, e.g., hepatitis B or C, cirrhosis of theliver, benign liver tumors, hemangiomas, hepatic adenomas, and focalnodular hyperplasias.

The term “inhibit” means to decrease or reduce an activityu, function,and/or amount as compared to a reference.

“Inhibiting cell growth or proliferation” means decreasing a cell'sgrowth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100%, and includes inducing cell death.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-Jag1 antibody” or “isolatednucleic acid encoding an anti-Notch2 antibody” refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “Jagged” or “Jag,” as used herein, refers to any native Jaggedfrom any vertebrate source, including mammals such as primates (e.g.humans) and rodents (e.g., mice and rats), unless otherwise indicated.The term encompasses “full-length,” unprocessed Jag as well as any formof Jagged that results from processing in the cell. The term alsoencompasses naturally occurring variants of Jagged, e.g., splicevariants or allelic variants.

The term “Jagged1” or “Jag1,” as used herein, refers to any native Jag1from any vertebrate source, including mammals such as primates (e.g.humans) and rodents (e.g., mice and rats), unless otherwise indicated.The term encompasses “full-length,” unprocessed Jag1 as well as any formof Jag1 that results from processing in the cell. The term alsoencompasses naturally occurring variants of Jag1, e.g., splice variantsor allelic variants. The amino acid sequence of an exemplary human andmurine Jag1 is shown in FIG. 9.

The term “level of expression” or “expression level” as used herein,refers to the amount of a polynucleotide, mRNA, or an amino acid productor protein in a biological sample.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “Notch,” as used herein, refers to any native Notch from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed Notch as well as any form ofNotch that results from processing in the cell. The term alsoencompasses naturally occurring variants of Notch, e.g., splice variantsor allelic variants.

The term “Notch2,” as used herein, refers to any native Notch2 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed Notch2 as well as any form ofNotch2 that results from processing in the cell. The term alsoencompasses naturally occurring variants of Notch2, e.g., splicevariants or allelic variants. The amino acid sequence of an exemplaryhuman Notch2 is shown in FIG. 8.

The term “Notch2 signaling inhibitor” refers to an agent that effectsdecreased Notch2 signaling, as defined above. Notch2 signalinginhibitors include Notch2-specific antagonists and Jag1-specificantagonists. A Notch2-specific antagonist decreases Notch2 signaling anddoes not significantly affect signaling by another Notch receptor(Notch1, 3, or 4 in mammals). Examples of Notch2-specific antagonistinclude agents that block Notch2 binding to a Notch2 ligand. AJag1-specific antagonist decreases Jag1-mediated signaling. Examples ofJag1-specific antagonists include agents that bind Notch2. Pan-Notchinhibitors, such as gamma secretase inhibitors, are explicitly excludedfrom Notch2 signaling inhibitors defined herein.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

“Relapsed” refers to the regression of the patient's illness back to itsformer diseased state, especially the return of symptoms following anapparent recovery or partial recovery. Unless otherwise indicted,relapsed state refers to the process of returning to or the return toillness before the previous treatment including, but not limited to,chemotherapies and stem cell transplantation treatments.

“Refractory” refers to the resistance or non-responsiveness of a diseaseor condition to a treatment (e.g., the number of neoplastic plasma cellsincreases even though treatment if given). Unless otherwise indicated,the term “refractory” refers a resistance or non-responsiveness to anyprevious treatment including, but not limited to, chemotherapies andstem cell transplantation treatments.

The term “secreted phosphoprotein1” or “SPP1” or “osteopontin,” as usedherein, refers to any native SPP1 from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed SPP1 as well as any form of SPP1 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of SPP1, e.g., splice variants or allelic variants. The aminoacid sequence of an exemplary human SPP1 is shown in FIG. 11.

The phrase “substantially reduced” or “substantially different,” as usedherein, refers to a sufficiently high degree of difference between twonumeric values (generally one associated with a molecule and the otherassociated with a reference/comparator molecule) such that one of skillin the art would consider the difference between the two values to be ofstatistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values).

The term “substantially similar” or “substantially the same,” as usedherein, refers to a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values).

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer,” “cancerous,” “cell proliferativedisorder,” “proliferative disorder” and “tumor” are not mutuallyexclusive as referred to herein.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on inhibition of Notchpathway components for the treatment of liver cancer.

In one aspect, methods of treating a liver cancer in an individual inneed thereof are provided, comprising the step of administering to theindividual an effective amount of a Notch2 signaling inhibitor. In someembodiments, the liver cancer is hepatocellular carcinoma, hepatoma,cholangiocarcinoma, hepatoblastoma, hepatic carcinoma, hepaticangiosarcoma, or metastatic liver cancer. In some embodiments, the livercancer is a refractory cancer.

Examples of Notch2 signaling inhibitors are known in the art and someare exemplified herein, including, but not limited to, soluble Notchreceptors, soluble Notch ligand variants, e.g., dominant negative ligandvariants, aptamers or oligopeptides that bind Notch2 or Jag1, organic orinorganic molecules that interfere specifically with Notch2 signaling,anti-Notch2 antagonist antibodies and anti-Jag1 antagonist antibodies.Examples of Notch2-specific antagonists include those described in U.S.Patent Application Publication No. US 2010/0111958 and Sjölund et al.,J. Clin. Invest. 118(1):217-228 (2008).

In certain embodiments, the Notch2 signaling inhibitor is an anti-Notch2antagonist antibody. In one such embodiment, the anti-Notch2 antagonistantibody is an antibody that binds to the extracellular domain of Notch2and effects decreased Notch2 signaling. In one such embodiment, theanti-Notch2 antagonist antibody is an anti-Notch2 NRR antibody.Anti-Notch2 NRR antibodies include, but are not limited to, anyanti-Notch2 NRR antibodies disclosed in International ApplicationPublication No. WO2010039832, which is expressly incorporated byreference herein in its entirety. Such antibodies include, but are notlimited to anti-Notch2 NRR antibodies that bind to the LNR-A and HD-Cdomains of Notch2 NRR. Exemplary anti-Notch2 NRR antibodies aremonoclonal antibodies designated herein as Antibody B, Antibody B-1,Antibody B-2, and Antibody B-3. Antibody B that binds to Notch2 NRR wasisolated from a phage library. That antibody was affinity matured togenerate Antibody B-1, Antibody B-2, and Antibody B-3. The sequences ofthe heavy chain and light chain hypervariable regions (HVRs) of AntibodyB, Antibody B-1, Antibody B-2, and Antibody B-3 are shown in FIGS. 12and 13, respectively. The sequences of the heavy and light chainvariable domains of Antibody B, Antibody B-1, Antibody B-2, and AntibodyB-3 are shown in FIGS. 14 and 15. Further embodiments of anti-Notch2 NRRantibodies are provided as follows.

In one aspect, an antagonist antibody that specifically binds to Notch2NRR is provided, wherein the antibody comprises at least one, two,three, four, five, or six HVRs selected from:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4;    -   (d) an HVR-L1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:9;    -   (e) an HVR-L2 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:14; and    -   (f) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.

In a further aspect, the antibody comprises an HVR-H3 comprising theamino acid sequence of SEQ ID NO:4 and at least one, two, three, four,or five HVRs selected from (a), (b), (d), (e), and (f) above. In afurther aspect, the antibody comprises (a), (b), (c), (d), (e), and (f)above. With respect to (a), (d), (e), and (f), any one or more of thefollowing embodiments are contemplated: HVR-H1 comprises an amino acidsequence of SEQ ID NO:1; HVR-L1 comprises an amino acid sequenceselected from SEQ ID NOs:5-8; HVR-L2 comprises an amino acid sequenceselected from SEQ ID NOs:10-13; and HVR-L3 comprises an amino acidsequence selected from SEQ ID NOs:15-18.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO:1, an HVR-H2 comprising the amino acidsequence of SEQ ID NO:3, and an HVR-H3 comprising the amino acidsequence of SEQ ID NO:4.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises an HVR-L1 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:9, anHVR-L2 comprising an amino acid sequence that conforms to the consensussequence of SEQ ID NO:14, and an HVR-L3 comprising an amino acidsequence that conforms to the consensus sequence of SEQ ID NO:19. Thefollowing embodiments are contemplated in any combination: HVR-L1comprises an amino acid sequence selected from SEQ ID NOs:5-8; HVR-L2comprises an amino acid sequence selected from SEQ ID NOs:10-13; andHVR-L3 comprises an amino acid sequence selected from SEQ ID NOs:15-18.In one embodiment, an antibody that binds to Notch2 NRR comprises anHVR-L1 comprising the amino acid sequence of SEQ ID NO:5; an HVR-L2comprising the amino acid sequence of SEQ ID NO:10; and an HVR-L3comprising the amino acid sequence of SEQ ID NO:15. In anotherembodiment, an antibody that binds to Notch2 NRR comprises an HVR-L1comprising the amino acid sequence of SEQ ID NO:6; an HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:11; and an HVR-L3 comprising theamino acid sequence of SEQ ID NO:16. In another embodiment, an antibodythat binds to Notch2 NRR comprises an HVR-L1 comprising the amino acidsequence of SEQ ID NO:7; an HVR-L2 comprising the amino acid sequence ofSEQ ID NO:12; and an HVR-L3 comprising the amino acid sequence of SEQ IDNO:17. In another embodiment, an antibody that binds to Notch2 NRRcomprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:8;an HVR-L2 comprising the amino acid sequence of SEQ ID NO:13; and anHVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

In one embodiment, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:10; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:15.    -   In another embodiment, an antibody that specifically binds to        Notch2 NRR is provided, wherein the antibody comprises:    -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:6;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:11; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:16.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:7;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:12; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:17.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:8;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:18.

In certain embodiments, any of the above antibodies further comprises atleast one framework selected from a VH subgroup III consensus frameworkand a VL subgroup I consensus framework.

In certain embodiments, an anti-Notch2 NRR antibody is affinity matured.For example, any one or more of the following substitutions in theindicated HVR positions (Kabat numbered) may be made in any combination:

-   -   in HVR-L1 (SEQ ID NO:5): S28N; I29N or V; S30R or K; S31R; Y32F    -   in HVR-L2 (SEQ ID NO:10): G50R; S53I or T; A55E    -   in HVR-L3 (SEQ ID NO:15): S93I or R; L96S or H        The specific antibodies disclosed herein, Antibody B as well as        affinity matured forms of Antibody B (B-1, B-2, and B-3), may        undergo further affinity maturation. Accordingly, affinity        matured forms of any of the antibodies described herein are        provided.

In certain embodiments, the Notch2 signaling inhibitor is an anti-Jag1antagonist antibody. In one such embodiment, the anti-Jag1 antagonistantibody is an antibody that binds to the extracellular domain of Jag1and effects decreased Notch2 signaling. In one such embodiment, theanti-Jag1 antagonist antibody is an anti-Jag1 EGF1-4 antibody. Anti-Jag1antibodies include, but are not limited to, any anti-Jag1 antibodiesdisclosed herein.

In further embodiments, the antibody is an anti-Jag1 antibody. In someembodiments, the antibody comprises at least one, two, three, four,five, or six HVRs selected from: (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:81; (b) HVR-H2 comprising an amino acid sequenceof SEQ ID NO:84; (c) HVR-H3 comprising an amino acid sequence of SEQ IDNO:87; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:88;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:89; and (f)HVR-L3 comprising an amino acid sequence of SEQ ID NO:92. In oneembodiment, the antibody comprises: (a) an HVR-H1 comprising the aminoacid sequence of SEQ ID NO:81; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO:82; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:85; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO:88; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:89; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:90.

In one embodiment, the antibody comprises: (a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO:81; (b) an HVR-H2 comprising the aminoacid sequence of SEQ ID NO:82; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:86; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO:88; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:89; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:91. In one embodiment, the antibody comprises: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:83; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:85; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:88; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:89; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:90.

In certain embodiments, an anti-Notch2 NRR antibody or anti-Jag1antibody having any of the above HVR sequences can further comprise anysuitable framework variable domain sequence, provided binding activityto Notch2 NRR and Jag1, respectively, is substantially retained. Incertain embodiments, an anti-Notch2 NRR antibody or anti-Jag1 antibodycomprises a human variable heavy (VH) consensus framework sequence, asin any of the VH consensus framework sequences shown in FIGS. 16A and16B. In one embodiment, the VH consensus framework sequence comprises ahuman subgroup III heavy chain framework consensus sequence, e.g., asshown in FIGS. 16A and 16B. In another embodiment, the VH consensusframework sequence comprises an “Acceptor 2” framework sequence, e.g.,as shown in FIGS. 16A and 16B. In a particular embodiment, the VHframework consensus sequence comprises FR1-FR4 of Acceptor 2B orAcceptor 2D, wherein the FR4 comprises SEQ ID NO:35 (FIGS. 16A and 16B),with the last residue of SEQ ID NO:35 (S11) optionally being substitutedwith alanine. In a further particular embodiment, the VH frameworkconsensus sequence comprises the sequences of SEQ ID NOs:50; 51; 57 or59; and 35, wherein S11 of SEQ ID NO:35 is optionally substituted withalanine.

In certain embodiments, an anti-Notch2 NRR antibody or anti-Jag1antibody having any of the above HVR sequences can further comprise ahuman variable light (VL) consensus framework sequence as shown in FIG.17. In one embodiment, the VL consensus framework sequence comprises ahuman VL kappa subgroup I consensus framework (κv1) sequence, e.g., asshown in FIG. 17. In another embodiment, the VL framework consensussequence comprises FR1-FR4 of huMAb4D5-8 as shown in FIG. 18 or 19. In aparticular embodiment, the VL framework consensus sequence comprises thesequences of SEQ ID NOs:60, 61, 62, and 63.

In another aspect, an anti-Notch2 NRR antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NOs:20. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-Notch2 NRRantibody comprising that sequence retains the ability to bind to Notch2NRR. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in the amino acid sequence of SEQID NOs:20. In certain embodiments, substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs). In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1,(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises a light chain variable domain(VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to an amino acid sequence selected from SEQ IDNOs:22-25. In certain embodiments, a VL sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-Notch2 NRRantibody comprising that sequence retains the ability to bind to Notch2NRR. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in an amino acid sequence selectedfrom SEQ ID NOs:22-25. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). In a particular embodiment, the VL comprises one, two or threeHVRs selected from (a) an HVR-L1 comprising an amino acid sequence thatconforms to the consensus sequence of SEQ ID NO:9; (b) an HVR-L2comprising an amino acid sequence that conforms to the consensussequence of SEQ ID NO:14; and (c) an HVR-L3 comprising an amino acidsequence that conforms to the consensus sequence of SEQ ID NO:19. In onesuch embodiment, the VL comprises one, two or three HVRs selected from(a) an HVR-L1 comprising an amino acid sequence selected from SEQ IDNOs:5-8; (b) an HVR-L2 comprising an amino acid sequence selected fromSEQ ID NOs:10-13; and (c) an HVR-L3 comprising an amino acid sequenceselected from SEQ ID NOs:15-18. In one such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:5; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:10; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:15. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:6; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:11; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:16. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:7; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:12; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:17. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:8; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:13; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:18.

In certain embodiments of the variant VH and VL sequences providedabove, substitutions, insertions, or deletions may occur within theHVRs. In such embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations that do not substantially reducebinding affinity may be made in HVRs. In certain instances, alterationsin HVRs may actually improve antibody affinity. Such alterations may bemade in HVR “hotspots” (i.e., residues encoded by codons that undergomutation at high frequency during the somatic maturation process) inorder to increase antibody affinity (See, e.g., Chowdhury, Methods Mol.Biol. 207:179-196, 2008.) In certain embodiments of the variant VH andVL sequences provided above, each HVR either is conserved (unaltered),or contains no more than a single amino acid substitution, insertion ordeletion.

In another aspect, an antibody that specifically binds Notch2 NRR isprovided, wherein the antibody comprises a VH as in any of theembodiments provided above, and a VL as in any of the embodimentsprovided above. In one embodiment, the antibody comprises a VH having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:20, and a VL having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:22. In one suchembodiment, the VH comprises one, two or three HVRs selected from: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:3, and (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:4, and the VLcomprises one, two or three HVRs selected from (a) an HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:5; (b) an HVR-L2 comprising theamino acid sequence of SEQ ID NO:10; and (c) an HVR-L3 comprising theamino acid sequence of SEQ ID NO:15. In a particular embodiment, theantibody comprises a VH comprising the amino acid sequence of SEQ IDNO:20, and a VL comprising the amino acid sequence of SEQ ID NO:22.

In another embodiment, an anti-Notch2 NRR antibody that specificallybinds Notch2 NRR comprises a VH having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:20, and a VL having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs:23-25. In one such embodiment, the VHcomprises one, two or three HVRs selected from: (a) an HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:1, (b) an HVR-H2 comprising theamino acid sequence of SEQ ID NO:3, and (c) an HVR-H3 comprising theamino acid sequence of SEQ ID NO:4, and the VL comprises one, two orthree HVRs selected from (a) an HVR-L1 comprising an amino acid sequenceselected from SEQ ID NOs:6-8; (b) an HVR-L2 comprising an amino acidsequence selected from SEQ ID NOs:11-13; and (c) an HVR-L3 comprising anamino acid sequence selected from SEQ ID NOs:16-18. In particularembodiments, the antibody comprises a VH comprising the amino acidsequence of SEQ ID NO:20 and a VL comprising an amino acid sequenceselected from SEQ ID NOs:23-25.

In another aspect, an antibody that specifically binds Jag1 is provided,wherein the antibody comprises a VH as in any of the embodimentsprovided above, and a VL as in any of the embodiments provided above. Inone embodiment, the antibody comprises a VH having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID NOs:93-95, and a VL having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID NOs:96-98. Inone such embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81,(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:82, and(c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:85, andthe VL comprises one, two or three HVRs selected from (a) an HVR-L1comprising the amino acid sequence of SEQ ID NO:88; (b) an HVR-L2comprising the amino acid sequence of SEQ ID NO:89; and (c) an HVR-L3comprising the amino acid sequence of SEQ ID NO:90. In a particularembodiment, the antibody comprises a VH comprising the amino acidsequence of SEQ ID NO:93, and a VL comprising the amino acid sequence ofSEQ ID NO:96.

In another embodiment, an anti-Jag1 antibody that specifically bindsJag1 comprises a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO:94, and a VL having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO:97. In one such embodiment, the VH comprises one, two orthree HVRs selected from: (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO:81, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO:82, and (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:86, and the VL comprises one, two or three HVRsselected from (a) an HVR-L1 comprising the amino acid sequence of SEQ IDNO:88; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:89;and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:91. Inparticular embodiments, the antibody comprises a VH comprising the aminoacid sequence of SEQ ID NO:94 and a VL comprising the amino acidsequence of SEQ ID NO:97.

In another embodiment, an anti-Jag1 antibody that specifically bindsJag1 comprises a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO:95, and a VL having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID NO:98. In one such embodiment, the VH comprises one, two orthree HVRs selected from: (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO:81, (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO:83, and (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:85, and the VL comprises one, two or three HVRsselected from (a) an HVR-L1 comprising the amino acid sequence of SEQ IDNO:88; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:89;and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:90. Inparticular embodiments, the antibody comprises a VH comprising the aminoacid sequence of SEQ ID NO:95 and a VL comprising the amino acidsequence of SEQ ID NO:98.

In certain embodiments, an affinity-matured form of any of the aboveantibodies is provided. In further embodiments, a recombinant proteinthat specifically binds Notch2 NRR or Jag1 is provided, wherein therecombinant protein comprises an antigen binding site(s) of any of theabove antibodies. In one such embodiment, a recombinant proteincomprises any one or more of the HVRs provided above.

In certain embodiments, a polynucleotide encoding any of the aboveantibodies is provided. In one embodiment, a vector comprising thepolynucleotide is provided. In one embodiment, a host cell comprisingthe vector is provided. In one embodiment, the host cell is eukaryotic.In one embodiment, the host cell is a CHO cell. In one embodiment, amethod of making an anti-Notch2 NRR antibody is provided, wherein themethod comprises culturing the host cell under conditions suitable forexpression of the polynucleotide encoding the antibody, and isolatingthe antibody.

In another embodiment, an isolated antibody is provided that binds tothe same epitope as an antibody provided herein. In one embodiment, anisolated anti-Notch2 NRR antibody is provided that binds to the sameepitope as an antibody selected from Antibody B, Antibody B-1, AntibodyB-2, and Antibody B-3. In another embodiment, the invention provides ananti-Notch2 NRR antibody that competes for binding with an antibodyselected from Antibody B, Antibody B-1, Antibody B-2, and Antibody B-3.In another embodiment, an isolated antibody is provided that binds to atleast one domain selected from the LNR-A domain and the HD-C domain ofNotch2. In one such embodiment, the antibody binds to both the LNR-Adomain and the HD-C domain. In another such embodiment, the antibodyfurther binds to the LNR-B and/or HD-N domains.

In one embodiment, an isolated anti-Jag1 antibody is provided that bindsto the same epitope as an antibody selected from Antibody A, AntibodyA-1, and Antibody A-2. In another embodiment, the invention provides ananti-Jag1 antibody that competes for binding with an antibody selectedfrom Antibody A, Antibody A-1, and Antibody A-2. In another embodiment,an isolated antibody is provided that binds to at least one domainselected from the DSL domain and the EGF domain of Jag1. In one suchembodiment, the antibody binds to EGF1-4 of Jag1.

Any of the Notch2 signaling inhibitors provided herein may be used inthe methods described herein.

The invention also provides methods for selecting a therapeutictreatment for a patient having a liver cancer, the method comprisingdetermining expression of one or more of Notch2, Jag1 and SPP1 in asample obtained from the patient. In some embodiments, the patient isselected for treatment with a Notch2 signaling inhibitor if expressionof one or more of Notch2, Jag1 and SPP1 is detected in the patientsample. In some embodiments, elevated expression of one or more ofNotch2, Jag1 and Spp1 in the sample obtained from the patient, relativeto a control, identifies the patient as suitable for receiving treatmentwith a Notch2 signaling inhibitor, as described herein. In someembodiments, additional parameters, such as, e.g., examination by aphysician, histologic evaluation of a biopsy, determination of serumlevels characterizing the liver cancer, are employed to identify thepatient for receiving the Notch2 signaling inhibitor treatment.

In some embodiments, a sample or biopsy from the patient is analyzed formRNA expression of one or more of Notch2, Jag1 and Spp1 using methodswell known in the art, such as, e.g., quantitative PCR analysis, andcompared to expression of the same gene or genes in a biopsy obtainedfrom a control individual or compared to a reference value. In someembodiments, expression is determined using enzyme linked immunosorbentassay (ELISA). In some embodiments, a sample or biopsy from the patientis analyzed for Notch2 activation, for example by detection of theactivated form of Notch2 as described herein.

In one aspect, methods are provided for preventing liver cancer in anindividual at risk of having liver cancer, comprising the step ofadministering to the individual an effective amount of a Notch2signaling inhibitor. In some embodiments, the individual has a livercondition selected from the group consisting of hepatitis B or C,cirrhosis of the liver, benign liver tumors, hemangiomas, hepaticadenomas, and focal nodular hyperplasia. In some embodiments, the methodfurther comprises administering at least one additional therapeuticagent. Examples of additional therapeutic agents include growthinhibitory agents, such as cytotoxic agents, peptides, small-moleculesand antibodies.

In another aspect, methods are provided for inhibiting the growth of acell that expresses secreted phosphoprotein1 (SPP1), comprisingcontacting the cell with a Notch2 signaling inhibitor, therebyinhibiting growth of the cell. In one embodiment, SPP1 protein comprisesthe amino acid sequence shown in FIG. 11. In one embodiment, contactingthe cell with the Notch2 signaling inhibitor reduces SPP1 expression inthe cell. For example, contacting the cell with the Notch2 signalinginhibitor reduces SPP1 expression in the cell by at least about 50%,60%, 70%, 80%, 90%, or 90%. The expression of SPP1 mRNA or protein canbe determined by any method in the art. In some embodiments, the cell isa liver cancer cell. In some embodiments, the liver cancer cellexpresses EpCAM, AFP, AFP and EpCAM, Notch2, Jag1, Notch2 and Jag1,nuclear Notch2 ICD, Ras, Prom1, Spp1, FoxM1, Plk1, ccnb1, Aurkb, Wnt2,Axin2, or Glu1, or any combination thereof. In some embodiments,contacting the cell with the Notch2 signaling inhibitor results in adecrease in the expression in the cell of at least one of EpCAM, AFP,Notch2, Notch2 ICD, Jag1, Prom1, Spp1, FoxM1, Plk1, ccnb1 and Aurkb. Insome embodiments, administering the Notch2 signaling inhibitor resultsin an increase in the expression in the cell of at least one of Wnt2,Axin2 and Glu1. In some embodiments, expression is determined by RNAseq,microarray analysis, immunohistochemistry, enzyme-linked immunosorbentassay, and Western blotting.

In another aspect, methods are provided for therapeutically treating amammal having a liver cancer comprising cells that express an Spp1 geneencoding a peptide comprising an amino acid sequence having at least 90%identity to the polypeptide shown in FIG. 11, comprising administeringto the mammal a therapeutically effective amount of a Notch2 signalinginhibitor, thereby effectively treating the mammal.

In another aspect, methods are provided for treating or preventing aliver cell proliferative disorder associated with increased expressionor activity of a protein having at least 90% amino acid sequenceidentity to the polypeptide shown in FIG. 8C, comprising administeringto an individual in need of such treatment an effective amount of ananti-Jag1 antagonist antibody, thereby effectively treating orpreventing the liver cell proliferative disorder. In some embodiments,the cell proliferative disorder is a cancer, such as liver cancer. Insome embodiments the individual has a liver condition selected from thegroup consisting of hepatitis B or C, cirrhosis of the liver, benignliver tumors, hemangiomas, hepatic adenomas, and focal nodularhyperplasia.

In one aspect, methods are provided for reducing serum SPP1 proteinlevels in an individual, the method comprising administering to theindividual an effective amount of a Notch2 signaling inhibitor therebyreducing serum SPP1 levels in the individual. In some embodiments,reducing is relative to serum SPP1 levels in the individual prior toadministering the Notch2 signaling inhibitor. In some embodiments,reducing is relative to a reference level. In one embodiment, theindividual has a liver cancer. In one embodiment, the serum SPP1 proteinlevels prior to administering the Notch2 signaling inhibitor to theindividual are at least about 80 ng/ml. In certain embodiments, theserum SPP1 protein levels prior to administering the Notch2 signalinginhibitor to the individual are between about 80 ng/ml and about 500ng/ml, between about 86 ng/ml and about 250 ng/ml, between about 120ng/ml and about 170 ng/ml, or about 165 ng/ml. In some embodiments,administering the Notch2 signaling inhibitor to the individual resultsin serum SPP1 protein levels of less than 80 ng/ml. In specificembodiments, serum SPP1 protein levels prior to administering the Notch2signaling inhibitor are 24 hours prior to administering the Notch2signaling inhibitor. Serum SPP1 protein levels prior to or followingadministration of the Notch2 signaling inhibitor may be determined byany appropriate method, such as enzyme-linked immunosorbent assay. Inspecific embodiments, serum SPP1 protein levels are reduced about one,two, three, six or 12 month after administering the Notch2 signalinginhibitor.

In one aspect, methods are provided for therapeutically treating a livertumor in a mammal, wherein the growth of the liver tumor is at least inpart dependent upon a growth potentiating effect of Notch2 signaling,comprising contacting the tumor with an antibody that binds to Notch2 orJag1, thereby effectively treating the tumor. In one embodiment, bindingof the antibody to the tumor antagonizes the growth-potentiatingactivity of Notch2.

In one aspect, methods are provided for preventing recurrence of livercancer comprising administering to an individual who has been treatedfor liver cancer and who has elevated serum SPP1 protein levels aneffective amount of a Notch2 signaling inhibitor. In one embodiment, theserum SPP1 protein levels of the individual are at least about 80 ng/ml.In certain embodiments, the serum SPP1 protein levels prior toadministering the Notch2 signaling inhibitor to the individual arebetween about 80 ng/ml and about 500 ng/ml, between about 86 ng/ml andabout 250 ng/ml, between about 120 ng/ml and about 170 ng/ml, or about165 ng/ml. In some embodiments, administering the Notch2 signalinginhibitor to the individual results in serum SPP1 protein levels of lessthan 80 ng/ml.

In some aspects, methods are provided for treating an individual havinga liver cancer, comprises the steps of administering to the individual aNotch2 signaling inhibitor; and determining Notch2 signaling, wherein adecrease in Notch2 signaling following treatment, compared to Notch2signaling prior to treatment, is indicative of reduction of liver cancerin the individual. In some embodiments, Notch2 signaling is determinedby measuring Notch2 ICD nuclear localization, e.g., byimmunohistochemical analysis. In some embodiments, Notch2 signaling isdetermined by measuring expression of a gene selected from the groupconsisting of Notch2, Jag1, Hes and Hey1. In some embodiments, the livercancer is hepatocellular carcinoma, hepatoma, cholangiocarcinoma,hepatoblastoma, hepatic carcinoma, hepatic angiosarcoma, and metastaticliver cancer. In some embodiments, the Notch2 signaling inhibitor is ansiRNA, small-molecule inhibitor or antibody. In some embodiments, theantibody is an antagonist antibody, such as an anti-Notch2 antagonistantibody or an anti-Jag1 antagonist antibody.

In some aspects, methods for inhibiting cellular proliferationcomprising treating mammalian liver cancer cells with an antibody toNotch2 or Jag1, whereby proliferation of the liver cancer cell isinhibited. In certain embodiments, the antibody is an anti-Notch2 oranti-Jag1 antagonist antibody is as described herein. In certainembodiments, the antibody is a human, humanized, or chimeric antibody.In certain embodiments, any of the antibodies of the above embodimentsis an antibody fragment. In certain embodiments, the cells are in apatient. In certain embodiments, the cells are in a culture medium.

Notch2 signaling inhibitors of the invention, such as anti-Notch2 andanti-Jag1 antibodies, can be used either alone or in combination withother agents in a therapy. For instance, an antibody of the inventionmay be co-administered with at least one additional therapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antagonist of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Notch2 signaling inhibitors of theinvention can also be used in combination with radiation therapy.

The antagonist can be administered to a human patient by any knownmethod, such as intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. TheNotch2 signaling inhibitor might be administered as a protein or as anucleic acid encoding a protein (see, for example, InternationalApplication Publication No. WO96/07321). Other therapeutic regimens maybe combined with the administration of the Notch2 signaling inhibitor.The combined administration includes co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. In some embodiments, such combined therapyresults in a synergistic therapeutic effect.

The dosage and mode of administration will be chosen by the physicianaccording to known criteria. The appropriate dosage of antibody, orother Notch2 signaling inhibitor, will depend on the type of disease tobe treated, the severity and course of the disease, whether theantibody, oligopeptide or organic molecule is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the Notch2 signaling inhibitor, and thediscretion of the attending physician. The Notch2 signaling inhibitorcan be administered to the patient at one time or over a series oftreatments.

Success of treatment of liver cancer can be monitored by assessingparameters of liver function and recovery. Such parameters include, butare not limited to, improved liver function tests, (e.g., assessingserum albumin, bilirubin, bile acids, total protein, clotting times),liver enzymes (e.g., alanine transaminase, aspartate transaminase,alkaline phosphatase, gamma glutamyl transpeptidase), histologicappearance (e.g., needle biopsy showing improved hepatic architecture),and imaging modalities (e.g., ultrasound, magnetic resonance imaging forfibrosis and liver size). Success of treatment can also be monitored bymeasuring serum levels of SPP1 protein, wherein a decrease in serumlevels in a treated patient, compared to pre-treatment levels, indicatesuccessful treatment.

In a further aspect, a Notch2 signaling inhibitor is an antibody used inany of the above embodiments that incorporates any of the features,singly or in combination, as described in Sections 1-7 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CMS chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CMS, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for Jag1 and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for Notch2 andthe other is for any other antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of Jag1. In certainembodiments, bispecific antibodies may bind to two different epitopes ofNotch2. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express Jag1 and/or Notch2. Bispecific antibodiescan be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to Jag1 or Notch2,as well as another, different antigen (see, US 2008/0069820, forexample).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinityAffinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-Jag1 antibody described herein isprovided. In one embodiment, isolated nucleic acid encoding ananti-Notch2 antibody described herein is provided. Such nucleic acid mayencode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychains of the antibody). In a further embodiment, one or more vectors(e.g., expression vectors) comprising such nucleic acid are provided. Ina further embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an anti-Jag1 antibody is provided,wherein the method comprises culturing a host cell comprising a nucleicacid encoding the antibody, as provided above, under conditions suitablefor expression of the antibody, and optionally recovering the antibodyfrom the host cell (or host cell culture medium). In one embodiment, amethod of making an anti-Notch2 antibody is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theantibody, as provided above, under conditions suitable for expression ofthe antibody, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of an anti-Jag1 antibody or an anti-Notch2antibody, nucleic acid encoding an antibody, e.g., as described above,is isolated and inserted into one or more vectors for further cloningand/or expression in a host cell. Such nucleic acid may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

The antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with Antibody A, A-1 or A-2 for binding to Jag1.In certain embodiments, such a competing antibody binds to the sameepitope (e.g., a linear or a conformational epitope) that is bound byAntibody A, A-1 or A-2. In another aspect, competition assays may beused to identify an antibody that competes with Antibody B, B-1, B-2 orB-3 for binding to Notch2. In certain embodiments, such a competingantibody binds to the same epitope (e.g., a linear or a conformationalepitope) that is bound by Antibody B, B-1, B-2 or B-3. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized Jag1 is incubated in asolution comprising a first labeled antibody that binds to Jag1 (e.g.,Antibody A, A-1 or A-2) and a second unlabeled antibody that is beingtested for its ability to compete with the first antibody for binding toJag1. The second antibody may be present in a hybridoma supernatant. Asa control, immobilized Jag1 is incubated in a solution comprising thefirst labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto Jag1, excess unbound antibody is removed, and the amount of labelassociated with immobilized Jag1 is measured. If the amount of labelassociated with immobilized Jag1 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding toJag1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). In anotherexemplary competition assay, immobilized Notch2 is incubated in asolution comprising a first labeled antibody that binds to Notch2 (e.g.,Antibody B, B-1, B-2 or B3) and a second unlabeled antibody that isbeing tested for its ability to compete with the first antibody forbinding to Notch2. The second antibody may be present in a hybridomasupernatant. As a control, immobilized Notch2 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to Notch2, excess unbound antibody is removed, andthe amount of label associated with immobilized Notch2 is measured. Ifthe amount of label associated with immobilized Jag1 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to Notch2.

2. Activity Assays

In one aspect, assays are provided for identifying Notch2 signalinginhibitor antibodies, such as anti-Jag1 antibodies and anti-Notch2antibodies, having biological activity. Biological activity may include,e.g., inhibition or reduction of Notch2 activity, e.g., Notch2signaling, inhibition or reduction of Jag1-mediated Notch signaling,e.g., Jag1-mediated Notch2 signaling. Antibodies having such biologicalactivity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. In certain embodiments, an antibody of theinvention is tested for its ability to inhibit reduce Spp1 expression.An exemplary assay is provided in the Examples. In certain otherembodiments, an antibody of the invention is tested for its ability toinhibit expression of a reporter gene that is responsive to Notch2signaling. In certain other embodiments, an antibody of the invention istested for its ability to inhibit expression of a reporter gene that isresponsive to Jag1-mediated signaling, e.g., Jag1-mediated Notch2signaling. In one exemplary assay, NIH-3T3 cells stably transfected withNotch 2 or transiently transfected with plasmids containing other Notchreceptors are co-transfected with a Notch-responsive TP-I (12X CSL)Firefly luciferase reporter and a constitutively active RenillaLuciferase reporter (pRL-CMV, Promega) to control for transfectionefficiency. Cells are allowed to recover from the transfection from 6hours to overnight. Treatments of antibodies and NIH-3T3 cells stablytransfected with ligand are used to stimulate the receptor cells. After20 hours, firefly and Renilla luciferase are measured with Dual GIoLuciferase Assay system (Promega). Replicates are analyzed for eachcondition by dividing the Firefly signal by the Renilla signal tocontrol for transfection efficiency. The mean and standard deviation arecalculated and values are normalized to calculated values for co-culturestimulated with NIH-3T3 cells without ligand transfected.

In certain embodiments, an antibody of the invention is tested for itsability to inhibit cell growth or proliferation in vitro. Assays forinhibition of cell growth or proliferation are well known in the art.Certain assays for cell proliferation, exemplified by the “cell killing”assays described herein, measure cell viability. One such assay is theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

Another assay for cell proliferation is the “MTT” assay, a colorimetricassay that measures the oxidation of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazanby mitochondrial reductase. Like the CellTiter-Glo™ assay, this assayindicates the number of metabolically active cells present in a cellculture. See, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhanget al. (2005) Cancer Res. 65:3877-3882.

In one aspect, an antibody of the invention is tested for its ability toinduce cell death in vitro. Assays for induction of cell death are wellknown in the art. In some embodiments, such assays measure, e.g., lossof membrane integrity as indicated by uptake of propidium iodide (PI),trypan blue (see Moore et al. (1995) Cytotechnology, 17:1-11), or 7AAD.In an exemplary PI uptake assay, cells are cultured in Dulbecco'sModified Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10%heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay isperformed in the absence of complement and immune effector cells. Cellsare seeded at a density of 3×10⁶ per dish in 100×20 mm dishes andallowed to attach overnight. The medium is removed and replaced withfresh medium alone or medium containing various concentrations of theantibody or immunoconjugate. The cells are incubated for a 3-day timeperiod. Following treatment, monolayers are washed with PBS and detachedby trypsinization. Cells are then centrifuged at 1200 rpm for 5 minutesat 4° C., the pellet resuspended in 3 ml cold Ca²⁺ binding buffer (10 mMHepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mmstrainer-capped 12×75 mm tubes (1 ml per tube, 3 tubes per treatmentgroup) for removal of cell clumps. Tubes then receive PI (10 μg/ml).Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). Antibodies which inducestatistically significant levels of cell death as determined by PIuptake are thus identified.

In one aspect, an antibody of the invention is tested for its ability toinduce apoptosis (programmed cell death) in vitro. An exemplary assayfor antibodies or immunconjugates that induce apoptosis is an annexinbinding assay. In an exemplary annexin binding assay, cells are culturedand seeded in dishes as discussed in the preceding paragraph. The mediumis removed and replaced with fresh medium alone or medium containing0.001 to 10 μg/ml of the antibody or immunoconjugate. Following athree-day incubation period, monolayers are washed with PBS and detachedby trypsinization. Cells are then centrifuged, resuspended in Ca²⁺binding buffer, and aliquoted into tubes as discussed in the precedingparagraph. Tubes then receive labeled annexin (e.g. annexin V-FITC) (1μg/ml). Samples are analyzed using a FACSCAN™ flow cytometer andFACSCONVERT™ CellQuest software (BD Biosciences). Antibodies that inducestatistically significant levels of annexin binding relative to controlare thus identified. Another exemplary assay for antibodies orimmunoconjugates that induce apoptosis is a histone DNA ELISAcolorimetric assay for detecting internucleosomal degradation of genomicDNA. Such an assay can be performed using, e.g., the Cell DeathDetection ELISA kit (Roche, Palo Alto, Calif.).

Cells for use in any of the above in vitro assays include cells or celllines that naturally express Notch2 and/or Jag1 or that have beenengineered to express Notch2 and/or Jag1. Such cells include tumor cellsthat overexpress Notch2 and/or Jag1 relative to normal cells of the sametissue origin. Such cells also include cell lines (including tumor celllines) that express Notch2 and/or Jag1 and cell lines that do notnormally express Notch2 and/or Jag1 but have been transfected withnucleic acid encoding Notch2 and/or Jag1. Exemplary cell lines providedherein for use in any of the above in vitro assays include NIH-3T3cells.

In one aspect, an antibody of the invention is tested for its ability toinhibit cell growth or proliferation in vivo. In certain embodiments, ananti-Jag1 antibody thereof is tested for its ability to inhibit tumorgrowth in vivo. In certain embodiments, an anti-Notch2 antibody thereofis tested for its ability to inhibit tumor growth in vivo. In vivo modelsystems, such as xenograft models, can be used for such testing. In anexemplary xenograft system, human tumor cells are introduced into asuitably immunocompromised non-human animal, e.g., an athymic “nude”mouse. An antibody of the invention is administered to the animal. Theability of the antibody to inhibit or decrease tumor growth is measured.In certain embodiments of the above xenograft system, the human tumorcells are tumor cells from a human patient. Such xenograft models arecommercially available from Oncotest GmbH (Frieberg, Germany). Incertain embodiments, the human tumor cells are cells from a human tumorcell line, such as HepG2, Hep3B, PCL/PRF/5, Snu387, Snu398, Snu423,Snu449, Snu475, Huh-7, HLE, HLF, JHH1, JHH4, JHH5 and JHH7. In certainembodiments, the human tumor cells are introduced into a suitablyimmunocompromised non-human animal by subcutaneous injection or bytransplantation into a suitable site, such as a mammary fat pad.

It is understood that any of the above assays may be carried out usingan immunoconjugate of the invention in place of or in addition to anNotch2 signaling inhibitor.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-Notch2antibody or anti-Jag1 antibody herein conjugated to one or morecytotoxic agents, such as chemotherapeutic agents or drugs, growthinhibitory agents, toxins (e.g., protein toxins, enzymatically activetoxins of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the antibodies provided herein is usefulfor detecting the presence of a Notch2 or fragment thereof, or a Jag1 orfragment thereof, in a biological sample. The term “detecting” as usedherein encompasses quantitative or qualitative detection. In certainembodiments, a biological sample comprises a cell or tissue, such ashepatocyte, liver cancer cell and liver tumor tissue.

In one embodiment, an anti-Notch2 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of Notch2 in a biological sample is provided. Incertain embodiments, a method of detecting the presence of Notch2intracellular domain (ICD) in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-Notch2 antibody as described herein under conditionspermissive for binding of the anti-Notch2 antibody to Notch2, anddetecting whether a complex is formed between the anti-Notch2 antibodyand Notch2. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-Notch2 antibody is used to select subjects eligiblefor therapy with an anti-Notch2 antibody, as described above, e.g. whereNotch2, in particular activated Notch2, is a biomarker for selection ofpatients.

In one embodiment, an anti-Jag1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of Jag1 in a biological sample is provided. Incertain embodiments, a method of detecting the presence of Jag1intracellular domain (ICD) in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-Jag1 antibody as described herein under conditionspermissive for binding of the anti-Jag1 antibody to Jag1, and detectingwhether a complex is formed between the anti-Jag1 antibody and Jag1.Such method may be an in vitro or in vivo method. In one embodiment, ananti-Jag1 antibody is used to select subjects eligible for therapy withan anti-Jag1 antibody, as described above, e.g. where Jag1 is abiomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include liver cancer, specifically, hepatocellular carcinoma.

In certain embodiments, labeled anti-Notch2 or anti-Jag1 antibodies areprovided. Labels include, but are not limited to, labels or moietiesthat are detected directly (such as fluorescent, chromophoric,electron-dense, chemiluminescent, and radioactive labels), as well asmoieties, such as enzymes or ligands, that are detected indirectly,e.g., through an enzymatic reaction or molecular interaction. Exemplarylabels include, but are not limited to, the radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates orfluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, luceriferases, e.g., firefly luciferase and bacterialluciferase (U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkalinephosphatase, β-galactosidase, glucoamylase, lysozyme, saccharideoxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-Notch2 or anti-Jag1 antibody asdescribed herein are prepared by mixing such antibody having the desireddegree of purity with one or more optional pharmaceutically acceptablecarriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.(1980)), in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide achemotherapeutic agent or another therapeutic antibody with theanti-Notch2 or anti-Jag1 antibody. In some embodiments, a formulationmay contain an anti-Notch2 antibody and an anti-Jag1 antibody. Suchactive ingredients are suitably present in combination in amounts thatare effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Compositions

Also provided herein are article of manufacture is provided comprising(a) a container; (b) a composition of matter contained within thecontainer comprising an anti-Notch2 antibody or an anti-Jagged1 antibodyand a carrier for the treatment of liver cancer; and (c) a label affixedto the container, or a package insert included with the container,referring to the use of the composition of matter for the therapeutictreatment of or the diagnostic detection of a liver cancer.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a chemotherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-Notch2 or Jag1 antibody and administration of an additionaltherapeutic agent occur within about one month, or within about one, twoor three weeks, or within about one, two, three, four, five, or sixdays, of each other. Antibodies of the invention can also be used incombination with radiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. For repeated administrations overseveral days or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-Notch2 or anti-Jag1 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. For example, an article of manufacture is providedcomprising (a) a container; (b) a composition of matter contained withinthe container comprising an anti-Notch2 antibody or an anti-Jagged1antibody and a carrier for the treatment of liver cancer; and (c) alabel affixed to the container, or a package insert included with thecontainer, referring to the use of the composition of matter for thetherapeutic treatment of or the diagnostic detection of a liver cancer.

Suitable containers include, for example, bottles, vials, syringes, IVsolution bags, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or combined with another composition effective fortreating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is aNotch2 signaling inhibitor, e.g., an anti-Notch2 antibody or anti-Jag1antibody. The label or package insert indicates that the composition isused for treating a proliferative disorder of the liver, such as livercancer. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aproliferative disorder of the liver, such as liver cancer.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-Notch2 or anti-Jag1 antibody.

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Murine Model of Liver Cancer

FVB-N mice (Charles River, Hollister) were subjected to hydrodynamictail vein injection of Ras, AKT, and Sleeping Beauty transposaseencoding plasmids as previously described (Ho et al., C., Hepatology55:833-845 (2012)). Briefly, 10 ug of pT3-CAGGS-NRasV12, 10 ug ofpT3-EF1A-AKT, and 0.8 ug CMV-SB (Ho et al., Hepatology 55:833-845(2012); Yant et al., Mol. Cell. Biol. 24:9239-9247 (2004)) were dilutedin approximately 2 mL of Saline Solution (0.9% NaCl) and injected intothe lateral tail vein of FVB-N mice in 5 to 8 seconds.

To model the development of liver cancer, mice were subjected tohydrodynamic tail vein injection of plasmids encoding oncogenic Ras andconstitutively active AKT along with Sleeping Beauty transposase, asdescribed above and as previously described (Ho et al., C., Hepatology55:833-845 (2012)). This model allows for efficient and stabletransfection of hepatocytes and reliable expression of the transfectedoncogenes. Within 5 weeks following hydrodynamic tail vein injection,mice developed numerous intrahepatic tumor masses. Much of the normalliver parenchyma was displaced by tumor epithelium and livers of thesemice expanded to as much as ten times their original size. Consistentwith previous reports, the tumors that developed in these mice compriseda wide spectrum of liver tumor types, including hepatocellular carcinoma(HCC) and cholangiocarcinoma (CC). Approximately 80% of tumor nodulesmet the histopathological criteria for hepatocellular carcinoma and 20%met histopathological criteria for identification as cholangiocarcinoma.

Each liver comprised dozens of tumors each of which expressed a givenmarker or combination of markers, such as AFP (FIG. 1A) and EpCAM (FIG.1C). Some tumors express both AFP and EpCAM (FIG. 1B). Tumors in thismodel varied in their expression of HCC- and CC-specific tumor markers(FIG. 1A). Expression of alpha-Fetoprotein (AFP), specific for HCC, wasdetected in about 12.8% of cells in tumor-bearing livers (FIG. 1D),compared to less than 1% in normal non-tumor-bearing livers. Expressionof EpCAM (FIG. 1B), a marker of cholangiocarcinoma, was less prevalentin this model, detected in an average of about 5% of all liver cells,compared to about 1% in normal non-tumor-bearing livers. Tumorsexpressing both HCC- and CC-specific markers were also observed (FIG.1C). These combined HCC-CC (cHCC-CC) tumors are characterized byparticularly aggressive clinical features (American-Cancer-Society.2012. Cancer Facts & Figures 2012. Atlanta: American Cancer Society) andshare gene expression patterns with liver progenitor cells (Coulouarn etal., Carcinogenesis 33:1791-1796 (2012)).

Example 2 Activation of Notch2 Signaling

To determine if liver cancer is associated with Notch2 activation,immunofluorescence analysis was performed on tumors arising in thelivers of AKT/Ras HTV mice. Liver tissues were embedded and frozen inO.C.T.™ freezing medium (TISSUE-TEK®), and cryosectioned at 8 μm.Sections were fixed in 4% paraformaldehyde (PFA) and stained usingprimary antibodies for Notch2 (Cell Signaling Technology), EpCAM(BioLegend), and AFP (R&D Systems). For image analysis,immunofluorescence-stained slides were scanned using an Ariol slidescanning system (Leica).

High levels of Notch2 activation, as determined by immunofluorescencedetection of nuclear Notch2, was observed in AFP⁺/EpCAM⁺ tumors (FIG.1E) and to a lesser extent in EpCAM⁺ tumors (FIG. 1E, FIG. 1F). Lessprominent staining for activated Notch2 was observed in other tumor celltypes (FIG. 1E).

Example 3 Generation of Antibodies

To determine if Notch2 signaling was important for driving thedevelopment or growth of liver cancer, specifically in double positivetumors, mice were subjected to hydrodynamic tail vain injection with theAKT/Ras construct as described in Example 1, and treated with ananti-Notch2 antibody, anti-Jag1 antibody or isotype control(anti-Ragweed) antibody.

a. Library Sorting and Screening to Identify Anti-Jagged 1 Antibodies

Human phage antibody libraries with synthetic diversities in theselected complementarity determining regions, mimicking the naturaldiversity of human IgG repertoire, were used for panning Fab fragmentsdisplayed on the surface of M13 bacteriophage particles. HumanJag1-DSL-EGF1-4 (FIG. 10) was used as antigen for library sorting. Nunc96 well Maxisorp immunoplates were coated overnight at 4° C. with targetantigen (10 μg/ml) and were blocked for 1 hour at room temperature withphage blocking buffer PBST (phosphate-buffered saline (PBS) and 1% (w/v)bovine serum albumin (BSA) and 0.05% (v/v) tween-20). Antibody phagelibraries VH (see, e.g., Lee et al., J. Immunol. Meth. 284:119-132,2004) and VH/VL (see Liang et al., JMB. 366: 815-829, 2007) were addedto antigen plates separately and incubated overnight at roomtemperature. The following day antigen-coated plates were washed tentimes with PBT (PBS with 0.05% Tween-20), and bound phage were elutedwith 50 mM HCl and 500 mM NaCl for 30 minutes and neutralized with anequal volume of 1 M Tris base (pH7.5). Recovered phages were amplifiedin E. coli XL-1 Blue cells. During the subsequent selection rounds,incubation of antibody phage with the antigen-coated plates was reducedto 2-3 hours, and the stringency of plate washing was graduallyincreased.

After 4 rounds of panning, significant enrichment was observed. 96clones were picked each from VH and VH/VL library sorting to determinewhether they specifically bound to human Jagged 1. The variable regionsof these clones were PCR sequenced to identify unique sequence clones.The affinities of phage antibodies were ranked using spot competitionELISA. The phage antibody IC50 values were further determined usingcompetitive phage-binding ELISA. Unique phage antibodies that bindspecifically to human Jagged 1 (and not Jagged 2), or to both Jagged 1and Jagged 2 were chosen and reformatted to full-length IgGs forevaluation in in vitro cell assays.

Clones of interest were reformatted into IgGs by cloning V_(L) and V_(H)regions of individual clones into a pRK mammalian cell expression vector(pRK.LPG3.HumanKappa) containing the human kappa constant domain, andexpression vector (pRK.LPG4.HumanHC) encoding the full-length human IgG1constant domain, respectively (Shields et al., J Biol Chem 2000; 276:6591-6604). The antibodies were then transiently expressed in mammalianCHO cells, and purified with a protein A column.

b. Construction of Libraries for Affinity Improvement of Clones Derivedfrom the V_(H) or V_(H)V_(L) Libraries

Phagemid pW0703, derived from phagemid pV0350-2b (Lee et al., J. Mol.Biol 340, 1073-1093 (2004), containing stop codon (TAA) in all CDR-L3positions and displaying monovalent Fab on the surface of M13bacteriophage) served as the library templates for grafting heavy chainvariable domains (V_(H)) of clones of interest from the V_(H) libraryfor affinity maturation. Both hard and soft randomization strategieswere used for affinity maturation. For hard randomization, one lightchain library with selected positions of the three light chain CDRs wasrandomized using amino acids designed to mimic natural human antibodiesand the designed DNA degeneracy was as described in Lee et al. (J. Mol.Biol 340, 1073-1093 (2004)). To achieve the soft randomizationconditions, which introduced the mutation rate of approximately 50% atthe selected positions, the mutagenic DNA was synthesized with70-10-10-10 mixtures of bases favoring the wild type nucleotides (Gallopet al., J. Med. Chem. 37:1233-1251 (1994)). For soft randomization,residues at positions 91-96 of CDR-L3, 30-33, 35 of CDR-H1, 50, 52,53-54, and 56 of CDR-H2, 95-98 of CDR-H3 were targeted; and threedifferent combinations of CDR loops, H1/L3, H2/L3, and H3/L3, wereselected for randomization.

For clones originated from V_(H)V_(L) library, phagemids containing 4stop codons (TAA) in each CDR and displaying monovalent Fab on thesurface of M13 bacteriophage were generated individually, and served asthe templates for kunkel mutagenesis for the construction of affinitymaturation libraries. Only soft randomization strategy was used forclones derived from V_(H)V_(L) library, as diversity of CDR-L3 was builtinto the naïve library. To achieve the soft randomization conditions,residues at positions 28-31 of CDR-L1, 50, 53-55 of CDR-L2, 91-96 ofCDR-L3, 30-35 of CDR-H1, 50-56 of CDR-H2, 95-100 of CDR-H3 weretargeted; and four different combinations of CDR loops, H1/L3*, H2/L3*,and H3/L3* and L1/L2/L3* (where * denotes the position of stop codons onthe template), were selected for randomization.

c. Phage Sorting Strategy to Generate Affinity Improvement

For affinity improvement selection, Jag1 antigens were firstbiotinylated under limiting reagent condition. Phage libraries weresubjected to one round of plate sorting and five rounds of solutionsorting with increasing stringency. For the first round of platesorting, 10 ug/ml antigen was first coated on Maxisorp plate andpreblocked with blocking buffer (1% BSA and 0.05% Tween20 in PBS). 3O.D./ml in blocking buffer of phage input were incubated to antigenplates for 3 hours. The wells were washed with PBS-0.05% Tween20 tentimes. Bound phage was eluted with 150 ul/well 50 mM HCl, 500 mM KCl for30 minutes, and subsequently neutralized by 50 ul/well of 1M Tris pH8,titered, and propagated for the next round. For subsequent rounds,panning of the phage libraries was done in solution phase, where phagelibrary was incubated with 100 nM biotinylated target protein (theconcentration is based on parental clone phage IC50 value) in 100 μlbuffer containing 1% Superblock (Pierce Biotechnology) and 0.05% Tween20for 2 hours at room temperature. The mixture was further diluted 10×with 1% Superblock, and 100 μl/well was applied to neutravidin-coatedwells (10 μg/ml) for 30 minutes at room temperature with gentle shaking.To determine background binding, control wells containing phage werecaptured on neutravidin-coated plates. Bound phage was then washed,eluted and propagated as described for first round. Five more rounds ofsolution sorting were carried out together with increasing selectionstringency. The first couple rounds of which is for on-rate selection bydecreasing biotinylated target protein concentration from 100 nM to 0.1nM, and the last two rounds of which is for off-rate selection by addingexcess amounts of non-biotinylated target protein (300 to 1000 foldmore) to compete off weaker binders at room temperature.

d. High Throughput Affinity Screening ELISA (Single Spot Competition)

Colonies were picked from the sixth round of screening. Colonies weregrown overnight at 37° C. in 150 μl/well of 2YT media with 50 μg/mlcarbenicillin and 1×10¹⁰/ml M13KO7 in 96-well plate (Falcon). From thesame plate, a colony of XL-1 infected parental phage was picked ascontrol. 96-well Nunc Maxisorp plates were coated with 100 μl/well ofeither Jag1 or Jag2 (0.5 μg/ml) in PBS at 4° C. overnight. The plateswere blocked with 150 μl of 1% BSA and 0.05% Tween20 in PBS 20 for 1hour.

35 μl of the phage supernatant was diluted with 75 μl of ELISA (enzymelinked immunosorbent assay) buffer (PBS with 0.5% BSA, 0.05% Tween20)with or without 5 nM Jag1 or Jag2 and let incubate for 1 hour at roomtemperature in an F plate (NUNC). 95 μl of mixture was transferred sideby side to the antigen coated plates. The plate was gently shaken for 15min and was washed ten times with PBS-0.05% Tween 20. The binding wasquantified by adding horseradish peroxidase (HRP)-conjugated anti-M13antibody in ELISA buffer (1:2500) and incubated for 30 minutes at roomtemperature. The plates were washed with PBS-0.05% Tween 20 ten times.Next, 100 μl/well of Peroxidase substrate was added to the well andincubated for 5 minutes at room temperature. The reaction was stopped byadding 100 μl 0.1M Phosphoric Acid (H₃PO₄) to each well and allowed toincubate for 5 minutes at room temperature. The O.D. (optical density)of the yellow color in each well was determined using a standard ELISAplate reader at 450 nm. In comparison to the OD_(450 nm) reduction (%)of the well of parental phage (100%), clones that had the OD_(450 nm)reduction (%) lower than 50% were picked for sequence analysis. Uniqueclones were selected for phage preparation to determine binding affinity(phage IC50) against Jag1 by comparison to respective parental clones.Then the most affinity-improved clones were reformatted into human IgG1for antibody production.

Parent antibody A and affinity matured antibodies A-1 and A-2specifically bound to human and murine Jag1, specifically, to Jag1DSL-EGF1-4, but not human or murine Jag 2.

The generation and characterization of certain anti-Notch2 NRRantibodies have been previously described. See PCT Application No.PCT/US09/059028.

Example 4 Treatment with Notch2 Signaling Inhibitor Reduces Tumor Burden

AKT/Ras HTV mice as described in Example 1 were treated with ananti-Notch2 antibody (15 mg/kg, 1×/week), anti-Jag1 antibody (10 mg/kg,1×/week) or isotype control antibody beginning the day of thehydrodynamic tail vain injection. Livers were imaged and weighed atnecropsy on a standard laboratory balance. Mice treated with the controlantibody developed a heavy tumor burden (FIG. 2A) five weeks followinghydrodynamic tail vain injection, with their livers increasing in sizeto about 8.9 g or approximately 31% of body weight, up from 1.2 g or5.8% of body weight (FIG. 2B) in normal, non-tumor-bearing mice.Treatment with either anti-Notch2 or anti-Jag1 antibody significantlyimpeded tumor development (FIGS. 2A and B; p<0.0001, n>8). Mice treatedwith anti-Notch2 antibody developed a significantly smaller tumor burdenwith their livers growing to an average of 5.1 g or 19.3% of body weight(FIG. 2B). Anti-Jag1 treatment had an even greater effect. In these micefinal liver weights averaged 4.3 g or 15.8% of body weight (FIG. 2B).

EpCAM⁺ and AFP⁺/EpCAM⁺ subsets of tumors (FIG. 1E), in which Notch2signaling was more highly activated than in EpCAM⁻ tumors as determinedby detection of nuclear Notch2 by immunofluorescence, were highlysusceptible to Notch2 pathway inhibition. EpCAM⁺ tumors (AFP⁻/EpCAM⁺ andAFP⁺/EpCAM⁺ tumors) were significantly reduced in area followingtreatment with either anti-Notch2 antibody or anti-Jag1 antibody (FIG.3A, FIG. 3B). Notch2 signaling was not as highly activated inAFP⁺/EpCAM⁻ tumors (FIG. 1E), suggesting that these tumors might not beaffected by Notch2 pathway inhibition. However, contrary to expectation,both anti-Notch2 treatment and anti-Jag1 treatment led to significantreduction in AFP⁺ tumor area (FIG. 3C). Taken together, these resultsdemonstrate that anti-Notch2 or anti-Jag1 antibody treatment blocks thedevelopment of a broad range of liver tumors in this model of livercancer. Successful treatment with anti-Notch2 and anti-Jag1 antibodiesresulted in reduction of overall tumor burden including both HCC-likeand cholangiocarcinoma-like tumors as indicated by significantreductions in AFP and EpCAM staining following anti-Notch2 and anti-Jag1antibody treatment.

Example 5 Inhibition of Notch1 and Notch3

Inhibition of Jag1 had a similar effect as inhibition of Notch2,suggesting that Jag1 and Notch2 are acting in the same pathway,specifically, that Jag1 acts as the ligand for Notch2 in supportingtumor formation. To determine if inhibition of other Notch receptorscould also reduce liver cancer formation or growth, mice were subjectedto hydrodynamic tail vain injection with the Ras/AKT construct asdescribed in Example 1 and treated with an anti-Notch1 antagonistantibody (10 mg/kg, lx/week) or an anti-Notch3 antagonist antibody (30mg/kg, 3×/week). Treatment with the anti-Notch1 antibody reduced liverweight in Ras/AKT HTV mice, compared to isotype controls, whiletreatment with an anti-Notch3 antibody did not significantly affectliver weight (FIG. 2D; p<0.02, n≥7). While anti-Notch2 or anti-Jag1treatment decreased the level of EpCAM transcript (FIG. 3E, p<0.005,n≥7), inhibition of Notch1 increased the cross-sectional area of EpCAMpositive tumors in the liver (FIG. 3D, FIG. 3E, p<0.02, n≥7) andincreased the expression of the cholangiocarcinoma marker Cytokeratin 19(CK19; FIG. 3F). Treatment with antagonist antibodies to anti-Notch1 oranti-Notch3 did not affect expression of Sox9, a liver progenitor celland progenitor cell-like tumor marker and cholangiocarcinoma-like tumormarker, while treatment with an anti-Notch2 or anti-Jag1 antibodydrastically decreased Sox9 at both the mRNA (FIG. 3G) and protein level(FIG. 3H).

Thus, treatment with anti-Notch1 or anti-Notch3 antibodies did notsignificantly decrease tumor burden. In fact, inhibition of Notch1caused an increase in the number and cross-sectional area occupied byEpCAM⁺ cholangiocarcinoma-like tumors. These results, taken togetherwith the observation of increased cholangiocarcinoma-like lesionsfollowing anti-Notch1 treatment, further supported the conclusion thatthere are opposing roles for Notch2 and Notch1 in liver cancer.

Example 6 Anti-Notch2 or Anti-Jag1 Antibody Treatment Reduces Notch2Activation

For immunohistochemical analysis, tissues were fixed in 10% NeutralBuffered Formalin, embedded in paraffin, and sectioned. 4 μm-thickformalin-fixed paraffin embedded human tissues were subject to staining.For Jag1 IHC staining, all steps were carried out on the VentanaDiscovery XT autostainer using Ventana detection reagents (VentanaMedical Systems, Tucson, Ariz.). Tissue sections were deparaffinized inEZPrep solution and pretreatment was done with Cell Conditioner 1 usingstandard incubation time. Tissue sections were then incubated with goatpolyclonal anti-Jag1 primary antibody (Santa Cruz Biotechnology Inc,Santa Cruz, Calif.; Cat# sc-6011) at 0.2 μg/ml for 32 minutes at roomtemperature followed by incubation with biotinylated rabbit anti-goatIgG antibody (Vector Labs, Burlingame, Calif.) at 7.5 μg/ml for 32minutes at room temperature. Both primary and secondary antibodies werediluted in 10% normal human serum (Jackson ImmunoResearch) in 3% BSA.The sections were subsequently incubated with anti-Rabbit OmniMAP-HRPreagent for 16 minutes at room temperature.

For Notch 2 IHC, all steps were carried out on the Ventana Discovery XTPlatform utilizing Ventana detection reagents (Ventana Medical Systems,Tucson, Ariz.). Sections were deparaffinized using EZ Prep andpretreatment was accomplished with Cell Conditioner 1 using standardincubation time. Sections were then incubated with rabbit monoclonalanti-Notch2 primary antibody (Clone D76A6, Cell Signaling Technologies,Beverley, Mass.) at 8 μg/ml for 60 minutes at 37° C., followed byincubation with anti-Rabbit OMNIMAP-HRP reagent for 32 minutes.

For Hes1, all steps were carried out on the Dako autostainer using DAKOwash buffer and DAKO Target Retrieval Solution. Sections weredeparaffinized, re-hydrated then incubated with DAKO Target RetrievalSolution at 99° C. for 20 min, quenched with 3% H2O2 for 4 minutes thenblocked with Avidin Biotin Blocking Kit (Vector Laboratories: cat#sp-2001). Sections were incubated 45 min at RT with 1 ug/ml anti-HES-1(clone NM1; MBL International) then 5 ug/ml secondary antibody Bt-Dkanti-Rat (JacksonImmunoResearch) 15 minutes followed byBiotinylated-Tyramide (1:50) in amplification diluent for 3 minutes.Sections were subsequently incubated with DAB and Hematoxylin IIreagents for chromogenic detection and counterstaining. Slides weredehydrated, cleared in xylenes and coverslipped. All Sections weresubsequently incubated with DAB and Hematoxylin II reagents forchromogenic detection and counterstaining. Slides were dehydrated,cleared in xylenes and coverslipped. For image analysis,immunohistochemistry slides were scanned using the Nanozoomer slidescanning system (Hamamatsu).

Quantitative real-time PCR (QRTPCR) was performed using the TaqManOne-Step RT-PCR Kit for one step reactions using the 7900 HT RT-PCRsystem (Applied Biosystems) with TaqMan probes (Applied Biosystems).Probes used were Notch1 (Mm00435245_m1, Hs01062014_m1), Notch2(Mm00803077_m1, Hs01050719_m1), Notch3 (Mm00435270_m1, Hs01128541_m1),Notch4 (Mm00440525), Jag1 (Mm00496902_m1), Jag2 (Mm01325629_m1), DLL1(Mm01279269_m1), DLL3 (Mm00432854_m1), DLL4 (Mm00444619), Hey1(Mm00516555_m1), CK19 (Mm00492980_m1) and Sox9 (Mm00448840_m1).

In keeping with the broader effect of Notch2 and Jag1 inhibition ontumor formation, Notch2 signaling, as determined by detection of nuclearNotch2 protein by immunofluorescence, was significantly reducedthroughout the tumor-bearing livers following treatment with eitheranti-Notch2 or anti-Jag1 (FIG. 4A; p<0.05, n≥7). This reduction appearedto be due to a direct effect on activation of the Notch2 protein, asoverall levels of Notch2 expression, as determined by quantitativeRT-PCR, did not change. Consistent with anti-Notch2 or anti-Jag1antibody treatment blocking Notch2 activation, immunostaining wasreduced for Hes1, a downstream transcriptional target of the Notch2signaling pathway. Control-treated Ras/AKT HTV livers showed high levelsof Hes1 staining in (FIG. 4D). However, treatment with anti-Notch2 oranti-Jag1 antibody significantly reduced Hes1 staining, consistent witheffective blockade of Notch2 signaling in the antibody-treated livers(FIG. 4C, FIG. 4D, p≤0.0001, n>10). Confirming that Notch2 signaling wasblocked, quantitative RT-PCR analysis revealed that the Notch pathwaytarget gene HeyL was also strongly decreased with either anti-Notch2 oranti-Jag1 antibody treatment (FIG. 4E, p<0.0001, n>7).

In each case, anti-Jag1 antibody treatment had the same effect asanti-Notch2 antibody treatment, further supporting the conclusion thatJag1 is acting primarily through, i.e. as a ligand for, Notch2 insupporting liver cancer formation and growth. Taken together, theseresults demonstrate that treatment with either anti-Notch2 or anti-Jag1antibody resulted in a reduction of Notch2 activation.

Example 7 Effect of Notch Inhibitory Antibodies on Expression of NotchSignaling Pathway Components

Mice subjected to AKT/Ras HTV were treated with antagonistic antibodiesto Notch1 (10 mg/kg, 1×/week), Notch2 (10 mg/kg, 2×/week), Notch3 (30mg/kg, 3×/week), or Jag1 (10 mg/kg, 1×/week). An anti-Ragweed antibodywas administered at 30 mg/kg, 3×/week as a negative control. After 5weeks, livers were harvested and quantitative real-time PCR wasperformed on the isolated RNA to determine the effect of treatment ontranscripts of Notch signaling pathway components. Inhibition ofindividual Notch family receptors did not lead to a compensatoryincrease in the expression of other Notch receptor family members.

As described above, inhibition of individual Notch receptors, Notch1,Notch2, and Notch3, had distinct effects in the AKT/Ras model of livertumor development. This result suggests that the individual Notchreceptors do not necessarily compensate for one another in liver cancer.To determine the effect of inhibition of individual Notch receptors andligands on expression of Notch receptor family members, expression wasassessed following treatment of mice injection with the AKT/Rasconstruct as described in Example 1 with either an anti-Notch1,anti-Notch2, anti-Notch3, anti-Jag1 antibody or isotype controlantibody. No increase in receptor transcript expression for any of theindividual Notch receptors was observed upon inhibition with any of thethree inhibitory antibodies (FIG. 5 A-C). On the contrary Notch2inhibition with a specific inhibitory antibody led to a significantdecrease in expression levels of both Notch3 and Notch4 (p<0.05, n≥7).Inhibition of Notch3 also led to a decrease in its own expression(p<0.005, n≥7). This is consistent with previous observations that bothNotch2 and Notch3 control the transcription of Notch3 (Wang et al., PLoSONE 7:e37365 (2012); Liu et al., Circulation Research 107:860-870(2010)). The effect of treatment with the antagonist antibodies was evengreater with respect to expression of Notch ligands Jag1 and DLL1.Notch2 inhibition led to a significant decrease in Jag1 expression (FIG.5E), likely as a result of the decrease in Jag1-expressingcholangiocarcinoma and progenitor-like tumors in these livers. Jag1inhibition had a similar effect, further confirming that Jag1 and Notch2are acting together in this tumor model. In contrast inhibition ofNotch1 significantly increased the expression of the Notch ligands Jag2and DLL1 (FIG. 5 F, FIG. 5G). This result might, at least in part, helpto explain the observed increase in cholangiocarcinoma-like lesions inlivers treated with an anti-Notch1 antagonist antibody.

In summary, a compensatory increase in expression of other Notchreceptors upon Notch2 inhibition was not observed, suggesting thatsuccessful treatment with anti-Notch2 or anti-Jag1 antibodies will notlead to resistance through upregulation of alternative Notch signalingcomponents. In fact, treatment with either Notch2 or Jag1 antagonistantibodies actually led to a decrease in Jag1 ligand expression, andtreatment with anti-Notch2 led to a decrease in Notch3 expression.

Example 8 Anti-Notch2 or Anti-Jag1 Antibody Treatment BlocksProgenitor-Like and Cholangiocarcinoma-Like Liver Tumor Growth

To address the mechanisms by which Notch2 inhibition is leading todecreased tumor burden, high throughput RNA sequencing analysis wasperformed. Mice subjected to AKT/Ras HTV were treated with antagonisticantibodies to Notch2 (10 mg/kg, 2×/week), Jag1 (10 mg/kg, 1×/week), oranti-Ragweed control (30 mg/kg, 3×/week). After 5 weeks, livers wereharvested and RNA was subjected to high-throughput sequencing.

Progenitor-cell and cholangiocarcinoma-like HCC expression signaturegene expression was down-regulated in tumor-bearing livers followinganti-Notch2 and anti-Jag1 antibody treatment. EpCAM and CK19 expressionwas significantly down-regulated, as was expression of CD133/Prom1 (FIG.6A and data not shown) and Spp1 (FIG. 6B), both markers of liverprogenitor cells. Because FoxM1 has previously been shown to play a rolein HCC proliferation in general (Xia et al., Carcinogenesis 33:2250-2259(2012)) and in the Ras/Akt model of liver cancer specifically (Ho etal., Hepatology 55:833-845 (2012)), we examined its expression and wereable to show that both Notch2 and Jag1 inhibition lead to a decrease inFoxM1 expression (FIG. 6C). Moreover, FoxM1 target genes (Laoukili etal., Nat Cell Biol 7:126-136 (2005)), PLK1 (FIG. 6D), Ccnb1 (FIG. 6E),and Aurkb (FIG. 6F) were also decreased in tumor-bearing livers treatedwith either anti-Notch2 or anti-Jag1 antibody compared to controls.Markers of Wnt signaling were increased upon Notch2 or Jag1 inhibition.Specifically, Wnt2 ligand was increased (FIG. 6G) as was the Wnt-pathwaytarget geneAxin2 (FIG. 6H). The expression of Glutamine synthetase(Glu1; FIG. 6I), a marker of a subset of terminally differentiatedhepatocytes, was also increased in tumor-bearing livers treated witheither anti-Notch2 or anti-Jag1 antibody compared to controls. Theseobservations are consistent with Notch2 inhibition inducing a decreasein tumor cell proliferation through downregulation of FoxM1 and inducinga differentiated hepatocyte fate through induction of Wnt-signaling(Boulter et al., Nat. Med. 18(4):572 (2012)). It is possible that Notch2signaling inhibition in liver cancer effects terminal differentiation oftumor cells into hepatocytes. Consistent with this hypothesis, Notch2and Jag1 inhibition led to an increase in transcriptional markers ofterminally differentiated hepatocytes as well as Wnt signaling, which isknown to be important in differentiation of hepatocytes from progenitorcells (Boulter et al., Nat. Med. 18(4):572 (2012)). Notch2 and Jag1inhibition may also be acting by decreasing proliferation and increasingtumor cell death.

Example 9 Expression and Activation of Notch2 in Human HepatocellularCarcinoma

Human HCC cell lines and primary human HCC tumors were analyzed usingquantitative reverse transcription polymerase chain reaction (qRT-PCR)for expression of Notch signaling components. HCC cell lines HepG2,Hep3B, PCL/PRF/5, Snu387, Snu398, Snu423, Snu449, Snu475 were acquiredfrom ATCC (Manassas, Va.). HCC cell lines Huh-7, HLE, HLF, JHH1, JHH4,JHH5, JHH7 were acquired from the Japanese Collection of BioresourcesCell Bank (Osaka, Japan). Whole stained tissue sections were analyzedusing Definiens software.

Notch2 was expressed at higher levels than either Notch1 or Notch3 in 15of 16 cultured HCC cell lines (FIG. 7A). In many cases, Notch2expression exceeded that of the other Notch family members by more than10 fold when expression was normalized to a reference gene (RPL19, FIG.7A). Consistent with this result, prominent expression of Notch2 wasobserved in 28 of 76 (37%) human primary HCC samples as determined byIHC. In 15 of these 28 human primary HCC samples (54%) Notch2 showedvarying degrees of nuclear localization (FIG. 7B) indicating Notch2pathway activation. Jag1 expression, evaluated by IHC, was observed in34 of 59 (57%) human primary HCC samples examined (FIG. 7B). Of the 56human primary HCC samples evaluated for both Notch2 and Jag1, 15 (27%)were found to have expression of both Notch2 and Jag1. Of those 15tissues with overlapping expression, 11 (73%) showed some degree ofNotch2 nuclear localization indicating active Notch2 signaling.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. A method of treating a liver cancer in anindividual in need thereof, comprising administering to the individualan effective amount of an anti-Notch2 antibody, wherein the liver cancercomprises cells that express alpha-fetoprotein (AFP) and/or EpCAM. 2.The method of claim 1, wherein the liver cancer is selected fromhepatocellular carcinoma, hepatoma, cholangiocarcinoma, hepatoblastoma,hepatic carcinoma, hepatic angiosarcoma, and metastatic liver cancer. 3.The method of claim 1, wherein the anti-Notch2 antibody is a monoclonalantibody.
 4. The method of claim 3, wherein the anti-Notch2 antibody isan IgG1 or IgG2a antibody.
 5. The method of claim 3, wherein theanti-Notch2 antibody is a chimeric antibody, a humanized antibody, or ahuman antibody.
 6. The method of claim 1, wherein the anti-Notch2antibody is an antibody fragment.
 7. The method of claim 1, wherein theanti-Notch2 antibody is an anti-Notch2 NRR antibody.
 8. The method ofclaim 1, wherein the anti-Notch2 antibody does not significantly bind toa Notch family member other than Notch2.
 9. The method of claim 1,wherein the anti-Notch2 antibody binds to mouse Notch2 NRR and humanNotch2 NRR.
 10. The method of claim 1, wherein the anti-Notch2 antibodybinds to a Notch2 NRR with a Kd of≤10 nM.
 11. The method of claim 1,wherein the anti-Notch2 antibody comprises heavy chain hypervariableregion 1 (HVR-H1) comprising the amino acid sequence of SEQ ID NO:1;heavy chain hypervariable region 2 (HVR-H2) comprising the amino acidsequence of SEQ ID NO:3; heavy chain hypervariable region 3 (HVR-H3)comprising the amino acid sequence of SEQ ID NO:4; light chainhypervariable region 1 (HVR-L1) comprising the amino acid sequence ofSEQ ID NO:9; light chain hypervariable region 2 (HVR-L2) comprising theamino acid sequence of SEQ ID NO:14; and light chain hypervariableregion 3 (HVR-L3) comprising the amino acid sequence of SEQ ID NO:19.12. The method of claim 1, wherein the anti-Notch2 antibody isconjugated to a cytotoxic agent.
 13. The method of claim 12, wherein thecytotoxic agent is selected from a toxin, an antibiotic, a radioactiveisotopes, and a nucleolytic enzyme.
 14. The method of claim 1, whereinthe liver cancer comprises cells that express at least one of Notch2 ,Jag1, Sox9, CK19, Ras, Prom1, Spp1, FoxM1, Plk1, ccnb1, Aurkb, Wnt2,Axin2, or Glu1.
 15. The method of claim 1, wherein the liver cancercomprises cells having nuclear Notch2 or activated Ras.
 16. The methodof claim 1, wherein the liver cancer comprises cells that express EpCAM,and wherein administering the anti-Notch2 antibody results in a decreasein EpCAM expression in the cells.
 17. The method of claim 1, wherein theliver cancer comprises cells that express AFP, and wherein administeringthe anti-Notch2 antibody results in a decrease in AFP expression in thecells.
 18. The method of claim 1, wherein administering the anti-Notch2antibody results in a decrease in expression, compared to expressionprior to administering the anti-Notch2 antibody, in cells of the livercancer of at least one of Prom1, Spp1, FoxM1, Plk1, ccnb1 and Aurkb. 19.The method of claim 1, wherein administering the anti-Notch2 antibodyresults in an increase in expression in cells of the liver cancer of atleast one of Wnt2, Axin2 and Glu1.
 20. The method of claim 1, whereinthe liver cancer is at least in part dependent upon a growthpotentiating effect of Notch2 signaling.